NZ728857B2 - Materials and methods relating to linkers for use in protein drug conjugates - Google Patents

Abstract

The present invention relates to protein drug conjugates, methods of manufacturing the same and their use in therapy. In particular, the present invention relates to protein drug conjugates comprising a globular protein, an improved linker and a drug for use in targeted drug delivery applications. The improved linker comprises a vinylpyridine motif.

Description

Materials and Methods Relating to Linkers for Use in Protein Drug Conjugates The present invention relates to protein drug conjugates, and methods of manufacturing the same. More especially, the present invention relates to providing a protein, linker and drug, for example a cytotoxin, to produce a protein drug conjugate. Additionally, the present invention provides an improved linker for use in protein drug conjugates and methods of introducing said linker into said protein drug ates. More ically, the protein drug conjugate may be an antibody drug conjugate (ADC) or an albumin and linker ning drug conjugate.

Protein drug conjugates, in particular antibody drug conjugates, are known to provide targeted delivery of highly potent drugs to specific tissue for ent.

More specifically, ADCs, which typically consist of an antibody linked via a chemical linker with labile bonds, to a biologically active cytotoxic or drug payload, are known for use in anticancer treatments. The targeted delivery offered by such n drug conjugates s from the ability of the antibody or the like to sensitively discriminate between healthy and diseased tissue, thus ensuring safe delivery of the highly potent drug.

As such antibodies, ally monoclonal antibodies (mAbs) are useful in targeted research, eutic, diagnostic and other biotechnology uses. More especially, mAbs are useful in the area of targeted treatments and medicaments. mAbs can be utilised by way of incorporation into a treatment by means of an Antibody Drug Conjugate (ADC). As discussed above, ADCs are a type of ive medicament believed to have particular utility in the ent of cancers, amongst other things, and are a relatively new technology. Generally, an ADC (for example) for treatment of a cancer will comprise a mAb linked to a cytotoxic payload or drug, which can provide a cell killing action. The connection between the mAb and the cytotoxic al (cytotoxin) will lly be provided by a chemically stable linker molecule. Two types of ADC linker systems are known in the art; ble and non-cleavable. For cleavable ADC linker systems there is a release mechanism which is preferably enzymatically driven, although alternative cleavable systems are known which are chemically labile, as detailed in Methods in Molecular Biology, Volume 1045, 2013, published by Humana Press. In non cleavable ADC linker systems the release route is due to mAb degradation by the machinery of the cell when the ADC is in use.

The market approval in Europe and the US of the ADCs is® (Seattle Genetics/Takeda Group) and Kadcyla® tech/Roche) has paved the way for increased ch into this ADC class of biotherapeutics.

Adcetris®, shown below (also known as brentuximab vedotin), is directed to the protein CD30, which is expressed in classical Hodgkin ma (HL) and systemic anaplastic large cell lymphoma (sALCL). Brentuximab n consists of the chimeric monoclonal dy brentuximab (cAC10, which targets the cell- membrane protein CD30) linked to a cathepsin cleavable linker (valine-citrulline), para-aminobenzylcarbamate spacer and the antimitotic agent monomethyl auristatin E (M MAE).

Kadcyla®, shown below, is trastuzumab emtansine, an ADC consisting of the monoclonal antibody trastuzumab (Trastuzumab) linked to the cytotoxic agent mertansine (DM1). Trastuzumab alone stops growth of cancer cells by binding to the HER2/neu receptor, s mertansine enters cells and prevents cell division by binding to tubulin; ultimately this binding action will result in cell apoptosis. The ate is typically abbreviated to T-DM1. Each molecule of trastuzumab emtansine consists of a single trastuzumab molecule bound to several les of mertansine, a cytotoxic maytansinoid ning a sulfhydryl group, h a crosslinking reagent known as SMCC. SMCC is succinimidyl trans(maleimidylmethyl) cyclohexanecarboxylate, a bi-functional cross linker, that contains two reactive functional groups, a succinimide ester and a maleimide. The succinimide group of SMCC reacts with the free amino group of a lysine residue in the trastuzumab le and the maleimide moiety of SMCC links to the free sulfhydryl group of mertansine, forming a covalent bond between the antibody and mertansine. \\ \ \ \ \\-j‘\~"" \ \, N "‘ \ i \' :' h \ c .

V)( ,\'. »‘ _\ § \§- §\ \ \--‘\ \ \\ .\. .v’ \; .s \.

.V» \- ,0 {3~ 6‘ W9 {‘"wk, While ADCs are well-known in the art, other protein drug conjugates comprising a globular protein with the ability to e targeted delivery of a drug payload are not as well-known. For example, albumin may offer a suitable alternative to antibodies in such protein drug conjugates.

Albumin consists of three structurally gous, largely helical domains (I, II and Ill), each consisting of two subdomains, A and B. Like other mammalian albumins, human albumin contains 17 disulfide bridges and a free thiol at Cys34, which es the largest fraction of free thiol in blood serum.

Albumin is the major protein responsible for the colloid osmotic pressure of the blood and functions as a transport vehicle for long chain fatty acids, bilirubin, metal ions such as copper (II) and nickel (ll), calcium and zinc. Albumin has an approximate serum half-life of 20 days, attributable to the size of the n (approx. 67 kDa) and also a consequence of recycling through neonatal Fc receptors (FcRn). The FcRn recycles n, as well as antibodies (more especially lgGs), in a pH-dependent non-competitive manner protecting both from protein degradation in the lysosome. Circulating albumin is internalised by endothelial cells where it binds to FcRn in the acidic environment of the early endosome (pH 6). This allows albumin to be recycled to the surface of the cell and released back into the blood ological pH). Albumin can be covalently ated to a cytotoxic drug or alternatively fused to a n-based therapeutic in order to increase bio-availability and improve drug cokinetics.

Solid tumours have a permeable vasculature and also poor lymphatic drainage which result in an accumulation and ion of macromolecules (>40 kDa) within the tumour interstitial fluid. Studies have demonstrated the retention of albumin in various malignant solid tumours. There is also emerging evidence for the ic binding of albumin by various receptors, some of which have been shown to be highly expressed on ant cells. Similarly, albumin is known to accumulate in inflamed joints of rheumatoid arthritis patients due to an increase in the permeability of the blood-joint barrier. A known application of albumin in drug delivery is liver ing using albumin conjugates containing galactose residues, which enter hepatocytes after interaction with the asialoglycoprotein receptor R), present in large amounts and high affinity only on these cells.

Accordingly, these targeting properties, as well as its availability, biodegradability, lack of toxicity and immunogenicity and aligned with its markedly long half-life, make albumin a le candidate for drug delivery. As such, albumin represents a suitable alternative to antibodies in n drug conjugate systems.

The successful delivery of a drug or cytotoxic d to the target tissue is dependent upon the ability of the linker to bind to the protein and drug and to remain bound until the protein drug conjugate reaches the target tissue.

Accordingly, there exists a need to provide ative and/or improved linkers for use in such protein drug conjugates which provide successful delivery.

Additionally, there exists a need to provide new and/or improved n drug conjugates, such as those comprising albumin, for safe and ive delivery of cytotoxic drugs or therapeutic peptides or polypeptides.

In one embodiment, there exists a need to provide alternative and ed ADCs, which can effectively provide cell apoptosis properties. More especially, there is a need to provide improved ADC linker molecules to ensure ive and effective conjugation during the manufacture of said ADCs, and provide effective cytotoxin d when in use. ingly, in a first aspect of the present invention, there is provided a protein drug conjugate comprising a globular protein, a linker and a drug. More specifically the present invention provides a protein drug conjugate comprising a linker capable of providing site specific ation via a lysine or cysteine group present on the protein, preferably a nitrogen containing heterocyclic aromatic ring comprising a vinyl substituent.

The present invention provides a linker for use in protein drug conjugates, with utility in g proteins and drugs, for example antibodies and cytotoxins to provide ADC molecules. The linker provides an improved targeted payload of the drug. onally or alternatively, the linker provides the protein drug conjugate with increased stability as compared to currently known linker molecules for use in ADCs, thus affording protein drug conjugates with improved safety properties and consequently ed tolerability profiles. More specifically, use of the presently disclosed linker molecule in ADCs provides increased potency of the ADC, when in use, over and above the equivalent unconjugated antibody.

In the context of the present invention, the term "globular protein" should be construed to cover any protein which has targeting capabilities and so has the ability to deliver a drug payload to a specific target . Accordingly, "globular proteins" include antibodies and fragments thereof, albumin and transferrin, as well as any other alternatives known for use in drug conjugates.

By "drug" it is meant any chemical nce which has a known biological effect on humans or s. In particular, the drug may be a pharmaceutical drug which is used in the treatment, cure, prevention or diagnosis of disease or to vise enhance physical or mental well-being. As will be iated, the drug may be a known drug which has obtained the necessary marketing authorisation or a novel drug which has not yet undergone testing or achieved marketing authorisation.

In one embodiment of the present invention, there is provided an dy drug conjugate comprising an antibody, a linker and a xin.

Preferably the antibody is an antibody, or fragment thereof, and more preferably a monoclonal antibody (mAb). The mAb can be selected from any known mAb.

The only limitation on the selection of the mAb for use in the present invention is that it must have a cysteine or lysine residue present to allow the conjugation reaction to take place. It is particularly preferred that the mAb contain a cysteine residue, as some especially red embodiments of the linker of the present invention show increased selectivity toward the thiol group present in the ne residue. However, in the case that a mAb is identified which does not ordinarily contain the preferred cysteine residue, s are known to the skilled person in the art to introduce cysteine in to mAbs.

According to an alternative embodiment of the present invention (as further detailed below), the antibody may be substituted for albumin.

All of the preferred embodiments discussed below, which may or may not be described in relation to the antibody containing ADCs or albumin drug conjugates, can be equally considered as preferred embodiments for the protein drug conjugate, as well as the ADC embodiment and the albumin drug conjugate embodiment. More especially, the description of the drug and linker below, should be considered to apply equally to the various embodiments of the n drug conjugates.

The drug may be a cytotoxic payload or a therapeutic peptide or ptide. In particular, where the protein is an antibody or a nt thereof and the n drug conjugate is an ADC, the drug is preferably a cytotoxin. Alternatively, where the protein is albumin, the drug may be a cytotoxin or a therapeutic peptide or polypeptide. ably the xin is a biologically active cytotoxic material. Most preferably the cytotoxin is an anticancer drug. The xin may be selected from the group comprising auristatins, maytansines, calicheamicin, anthracycline and the pyrrolobenzodiazepenes. More especially, the cytotoxin may be monomethyl auristatin E (MMAE), doxorubicin or mertansine (DM1). It should be apparent to the person skilled in the art that MMAE is also known as (S)-N- ((3R,4S,5S)((S)((1R,2R)(((1S,2R)hydroxyphenylpropan yl)amino)methoxymethyloxopropyl)pyrrolidinyl)methoxymethyl oxoheptanyl)-N,3-dimethyl((S)methyl (methylamino)butanamido)butanamide.

However, additionally or alternatively, the xin could also be selected from other known cytotoxins ing ricin subunits and other peptide based cytotoxic materials, although such materials are less commonly utilised in the field of the art.

Where the drug is a therapeutic peptide or polypetide, the peptide or polypeptide may be any peptide or polypeptide which has therapeutic properties, for example antinociceptive, antidiabetes, antitumor or antiviral activity.

Additionally, or alternatively, the drug preferably comprises an amine group, a thiol group, or a carboxylic acid group, as these types of groups provide ideal sites for conjugation of the drug with the linker of the present invention. ably the linker (also referred to herein as the linker molecule or linker group) es site specific conjugation via the lysine or cysteine group present on the protein, such as an antibody.

More preferably the linker of the present invention comprises a nitrogen containing heterocyclic aromatic ring comprising a vinyl substituent.

In very general terms, the linker can be considered to comprise of three parts; a vinyl group, a nitrogen containing cyclic ic ring, and a linker arm.

The linker arm can be varied to result in differing terminal groups to provide reaction sites for either the drug or protein, for example the dy of an ADC, to bind to the linker.

Preferably, the nitrogen containing heterocyclic aromatic ring is a pyridine, pyrimidine, imidazole or aziridine ring. More preferably, the nitrogen containing heterocyclic ic ring is a pyridine ring or a dine ring and most preferably, it is a pyridine ring. When pyridine or dine rings are employed, preferably the vinyl group is in the 2—position or tion relative to a nitrogen heteroatom. 4-vinylpyridines are particularly preferred as they have been found to provide commercially acceptable rates of reaction in certain circumstances, as sed further below.

Examples of the preferred linkers in accordance with the present invention include: \ NH; \ /' [11 [2] \ OH OH I \ N/ [31 [4] \ JOOL N [5] (31:3. 0 \ 0H [6] CF3 o NI \ OH ?/ 0 N/ OH N/ S\)J\OH o / o OH Ha" "\iN- N153" : N g ,2 - / \HO 0 \ I l / O N N3 OH o / o OH OH [18] Of the examples shown above, the 4-vinylpyridine structures are particularly preferred, and the structure shown as [13] is most especially preferred. 4- Vinylpyridine structures are preferred, as in certain conditions, they show an increased reactivity over and above equivalent lpyridine structures.

As will be appreciated by the skilled person, each of the structures exemplified above are provided predominately with a terminal carboxylic acid group on the linker arm. However, these ures may preferably be provided in a tuted form, such that the preferred vinylpyridine ure comprises a preferred poly-(alkylene glycol) group, most preferably a PEG molecule. The presence of a PEG group on the linker molecule arm has been found to be most preferable as it enhances reaction efficiency when seeking to obtain the required drug to protein ratio in the protein drug conjugate methods of manufacture.

Such preferred linker molecules include the following examples: PH PL? PLE F'Ld PLa PLl? F'Lll FLT-3 FL13 F'L‘lil o REG O NH Pact, AK x\ «N ? v Tl "X"? I / H )Lij % N \PEG \ O PL15 Asymmetric dual loaded PL13 o / o PEG PEG Symmetrical dual loaded PL-13 In a preferred embodiment of the present invention, the linker comprises a molecule having the general formula: x N \\ R3 (I) wherein: - X and Y are independently selected from CH or N.

- R1 is selected from; - (CH2)n R, or, - (Cl-l2)m—Z— R, or, - (CH2)m—Z CO()- R, or, - (CH2)n— C(O)— Z- R, or, - — Z— (CH2)n —C(O)—R, or, - (CH2)m— Z— (CH2)n —C(O)—Z—R, or, - (CH2)m- Z C—(O)— (CH2)n—Z—(CH2)n—C(O)—Z—R, or ' (CH2)n — CH(C02R)2, 0r ' (CH2)m— Z — (CH2)2CH(C02R)2, 0r ' (CH2)n —Z1.

- R2 and/or R3 may be selected from the same group of molecules as R1_ However, ably R2 and/or R3 are selected from hydrogen or an electron withdrawing group, such as halogen (F, Cl, or Br), -NOZ, - COZH, -COZR4, COR4, -CHO, -CN, -CF3, 4R5 where R4 and R5 are independently selected from hydrogen or C140 alkyl; or, - R2 and/or R3 may be selected from en, alkyl or phenyl, this is particularly preferred when the linker molecule comprises a pyridine ring, as the alternative electron withdrawing groups may have a negative effect on the reactivity of the linker; or - R2 and R3 together form a fused (hetero) aromatic ring substituent which may e, but is not limited to, an indole, indazole, benzimidazole, quinoline, isoquinoline, aziridine or a purine.

When R2 and/or R3 is selected to be from an alkyl group, a methyl group (CH3), or a tert-butyl 3C) is red. The presence of an alkyl group on the linker molecule is preferred as the presence of such a group increases the basicity of the nitrogen present in the ring structure and s in the linker structure having increased reactivity over and above equivalent linker molecules.

When R2 and/or R3 is selected from an electron withdrawing group, CF?> is preferred. The presence of CF?> increases the reactivity of the vinyl group t in the linker molecule, and is stable under physiological conditions.

Alternatively, it is contemplated that in some embodiments, it is possible to employ more extended fused heterocyclic aromatic ring systems such as an indole, indazole, benzimidazole, quinoline, isoquinoline, aziradine or a purine.

In the formulas given above, Z can be independently selected from NH, O, or S, Z1 is an N3 group or an OH group n can be any integer from O to 10. In some preferred embodiments n is a value from O and 5. m can be any r from O and 10. Preferably m is from O and 5, and most preferably m is from O and 3, as shown in the examples given above. Additionally, or alternatively, in linker molecules where the Z group following the (CH2)m group is an 0 group and R1 (and R2 and/or R3 where they are selected from the same group of les) is in the 2 or 6 position on the ring structure, then m is preferably at least 3, as the 0 group should be spaced from nitrogen of the ring structure. This is due to the reducing effect that the oxygen group has been found to have on the reactivity of the linker molecule structures. This effect is not felt when the oxygen group is further spaced from the ring ure. This situation is reflected in the exemplified structures above.

R may be a hydrogen (H), hydroxide (OH), amine or a poly-(alkylene glycol) group. Preferably R is a poly-(alkylene glycol), and most preferably it is a PEG. As will be appreciated by the skilled , when the R group is PEG, it may preferably be proceeded by a Z group in the form of NH, due to the reaction t of addition of the PEG. This option is detailed in the generic formulae of R1 given above. Generally, the poly- (alkylene glycol) le is directly covalently bonded to the R1 and/or R2 groups as shown in the formula above. The linker group arms may have different lengths to keep the poly-(alkylene glycol) molecule closer or further away from the protein, for example the antibody.

Preferably the drug, such as a cytotoxic, is bound to the linker R1 group. In cases where R2 and/or R3 are selected from the same group of molecules as R1, as described above, then it is possible that these groups will also bind to drugs, such as cytotoxins, to provide protein drug conjugate, such as ADCs, with multiple drugs present in their structures. Some embodiments of this type may be referred to as symmetrical g of the drug in the protein drug conjugate.

Additionally or alternatively, where R1, and optionally R2 and/or R3, are selected from (CH2)n— CH(COZR)2, or (CH2)m— Z — (CH2)2CH(COZR)2, i.e. where the linker arm is branched, then it is possible that the drugs bind to each terminating group of the linker arm. As such multiple drugs are present in the n drug conjugate and may be described as trical loading of the drug.

Preferably, the nitrogen containing heterocyclic aromatic ring is a substituted pyridine ring (X and Y are both CH) or a substituted pyrimidine ring (one of X and Y is CH and the other is N). Most preferably the nitrogen containing cyclic aromatic ring is a pyridine ring.

In one particularly red embodiment R1 is either; (CH2)n — C(O) — R, or, (CH2)n — C(O) — Z - R, or, (CH2)m — Z — (CH2)n — C(O) — R, or (CH2)m — Z — (CH2)n — C(O) — Z — R.

In a further embodiment of the present invention, it might be desirable to include an extender linker within the linker molecule described above. Such an extender linker may be necessary to alter solubility or immunogenic properties of the functionalising group. More especially such an extender linker will be useful in a cleavable ADC linker system, which will be described in more detail below. le extender linkers for utilisation in the present invention will be apparent to the skilled person in the art, and particularly red extender linkers are described in US Patent Number 7,659,241 and US Patent Number 5,609,105.

Most preferably the extender linker is an enzyme cleavable extender linker that comprises a collection of amino acids that can be cleaved by an ellular protease.

In a further embodiment of the present ion it might be desirable to provide the linker in a modified form, such that it comprises an ester group to facilitate binding of the linker to the protein of the protein drug conjugate. This aspect will be described further below.

Preferred linkers in accordance with the present invention may be represented by the following formulae, N (ll);or R1 N 2 (III) wherein: R1 is selected from; - (CH2)n -C(O)-R, or - (H2)m—Z— R, or I (CH2)m_Z CO() R, or - (CH2)n— C(O)— Z- R, or - (CH2)m— 2— (CH2) —C(O)—R, or ' (CH2)m— Z— (CH2)n C—(O)—Z—R, or - (CH2)m- Z C—(O)— —Z—(CH2)n—C(O)—Z—R, or ' — CH(C02R)2,0 - (CH2)m— Z— (CH2)2CH(COZR)2,o . (CH2)n—Z1 - R2 may be selected from the same group of molecules as R1_ However, preferably R2 is selected from hydrogen or an electron withdrawing group, such as halogen (F, Cl, or Br), -NOZ, -COZH, - COZR4, COR4, -CHO, -CN, -CF3, -SOZNR4R5 where R4 and R5 are independently selected from hydrogen or C140 alkyl; or, - R2 may be selected from hydrogen, alkyl or phenyl, this is particularly preferred as the ative electron withdrawing groups may have a negative effect on the reactivity of the linker.

When R2 is selected to be from an alkyl group, a methyl group (CH3), or a tert- butyl 3C) is preferred. The presence of an alkyl group on the linker molecule is preferred as the presence of such a group increases the basicity of the nitrogen present in the ring structure and results in the linker structure having increased reactivity over and above lent linker molecules.

When R2 is selected from an electron withdrawing group, CF?> is preferred. The presence of CF?> increases the reactivity of the vinyl group present in the linker molecule, and is stable under physiological conditions.

In the formulas given above, - Z can be independently selected from NH, O, or S, - Z1 is independently selected from N3 or OH, - n can be any integer from O to 10. In some red embodiments n is a value from O and 5, - m can be any integer from O and 10. ably m is from O and 5, and most preferably m is from O and 3, as shown in the examples given above. Additionally, or alternatively, in linker molecules of formula (III) where the Z group following the (CH2)m group is an 0 group, then m is preferably at least 3, as the 0 group should be spaced from the en of the ring structure. This is due to the reducing effect that the oxygen group has been found to have on the reactivity of the linker molecule structures. This effect is not felt when the oxygen group is further spaced from the ring structure. This ion is reflected in the exemplified structures above, - R is a hydrogen (H), hydroxide (OH), amine or a poly-(alkylene glycol) group. Preferably R is a poly-(alkylene glycol), and most preferably it is a PEG. As will be appreciated by the skilled person, when the R group is PEG, it may preferably be proceeded by a Z group in the form of NH, due to the reaction product of addition of the PEG. This option is ed in the generic formulae of R1 given above. Generally, the poly-(alkylene ) molecule is directly covalently bonded to the R1 and/or R2 groups as shown in the formula above. The linker group arm may have different lengths to keep the poly-(alkylene glycol) molecule closer or further away from the protein, for example the antibody.

Preferably the drug, for example a cytotoxic, is bound to the linker R1 group. In cases where R2 is selected from the same group of molecules as R1, as described above, then it is le that these groups will also bind to drugs, e.g. cytotoxins, to provide protein drug conjugates, e.g. ADCs, with multiple drugs present in their structures. Some embodiments of this type may be referred to as symmetrical loading of the drug in the protein drug conjugate.

Additionally or alternatively, where R1, and optionally R2, are ed from (CH2)n — CH(COZR)2, or (CH2)m — Z — (CH2)2CH(COZR)2, i.e. where the linker arm is branched, then it is possible that the drugs bind to each terminating group of the linker arm. As such multiple drugs are present in the protein drug ate and may be bed as asymmetrical loading of the drug.

In one ment of the present ion, the linker is represented by the formula (III).

Additionally, or alternatively, where a poly(alkylene glycol) group is present, the basic poly-(alkylene glycol) structure may be provided with one or more reactive functional groups such as y, amine, carboxylic acid, alkyl halide, azide, succinimidyl, or thiol groups to facilitate the reaction of the poly-(alkylene glycol) molecule with the drug or protein. Particularly preferred poly-(alkylene glycol) molecules e those substituted at one or more hydroxyl positions with a chemical group, such as an alkyl group having between one and four carbon atoms. The most preferred poly-(alkylene glycol) molecules for use in accordance with the present invention are polyethylene glycol ("PEG") molecules, as mentioned above, although the skilled person would be able to use the techniques sed herein in conjunction with other poly-(alkylene ) molecules, such as opylene glycol or polyethylene-polypropylene glycol copolymers. Poly-(alkylene glycol) molecules, including PEGs, typically have molecular weights between about 200 Da and about 80 kDa. Preferred poly- (alkylene glycol) molecules have molecular weights between about 200 Da and 1 kDa. Poly-(alkylene glycol) molecules that may be used in accordance with the t invention are well known in the art and publicly ble, for example from commercially available sources such as Sigma Aldrich.

In a further aspect of the present invention there is provided non-cleavable and/or cleavable protein drug conjugate (e.g. ADC) linker systems.

A non-cleavable protein drug conjugate (e.g. ADC) linker system may be provided, which comprises a protein (e.g. an antibody), a linker, and a drug (e.g. a cytotoxin). In one system of this type, conjugation of an amine group on the dy is achieved via an activated ester present on the linker, which then in turn binds to the cytotoxin via the vinyl group. In this non-cleavable system the antibody is preferably a mAb. Additionally, the linker preferably comprises a poly-(alkylene ) molecule, most preferably a polyethylene glycol (PEG) molecule and the cytotoxic contains a thiol. As will be appreciated, in the above bed system, the dy is provided as an example of a protein and may be substituted for albumin and the cytotoxin is provided as an example of a drug and may be substituted for a therapeutic peptide or polypeptide.

In an alternative system of this type, conjugation of a thiol group on the antibody is achieved via the vinyl group of the linker, which in turn binds to a drug via a poly(alkylene glycol) group present on the linker arm.

In an alternative embodiment, there is provided a cleavable protein drug conjugate (e.g. ADC) linker system, which comprises a protein (e.g. an antibody), linker, extender linker and a drug (e.g. a cytotoxin).

In a cleavable ADC linker system, the vinyl substituent of the linker molecule is especially suited to reacting with one or more thiol groups that are naturally t, or have been introduced into, the antibody (for example by employing a thiol group of one or more cysteine residues present on the antibody). The linker side-arm is then ted to the extender linker, preferably via the poly- (alkylene ) molecule; the extender linker provides the "cleavable" function to the ADC in this embodiment. The cytotoxin is then bound to the linker via the extender linker. Preferably, the antibody is a mAb. More preferably, the linker comprises a poly-(alkylene glycol) molecule, most preferably a polyethylene glycol (PEG) le. It is preferred that the extender linker is enzyme cleavable, as described in further detail above. As will be appreciated, in the above described system, the antibody is provided as an example of a protein and may be substituted for albumin and the cytotoxin is provided as an example of a drug and may be tuted for a therapeutic peptide or polypeptide.

It should be tood that in the t application the term "cleavable" is used to encompass n drug conjugates (e.g. ADCs) which are able to self immolate or may be manipulated to release their drug (e.g. cytotoxic) payload.

Use of the term is intended to distinguish such protein drug conjugates (e.g.

ADCs) from those "non-cleavable" n drug conjugates (e.g. ADCs) which are understood to only release their drug (e.g. cytotoxic) payload once present in a target cell.

It should be understood that the preferable features of the protein (e.g. antibody), linker and drug (e.g. xin) for utilising in this aspect of the invention are as described above in relation to the first aspect. More especially, the description of the linker molecule and its preferable features are particularly suited for use in said cleavable and non-cleavable s.

Although it is preferred that the protein (e.g. dy) utilised in the present invention comprise at least one or more thiol containing ne group, it is also plated that the protein (e.g. antibody) may contain one or more lysine group. When this is the case it is contemplated that the skilled person may prefer to attach the linker of the present invention to a lysine group as an ative to a cysteine group. In this situation it is preferred to modify the linker molecule such that it is an activated ester form of the linker. In this embodiment the ester group of the modified linker is able to bind to the lysine present on the protein (e.g. antibody) via the linker arm. In this embodiment, the n (e.g. the antibody) will bind via a lysine group to the ester modified , and in turn this will bind to the drug (i.e. the cytotoxin) via the vinyl group. This modified linker may comprise a alkylene glycol) molecule, most preferably a hylene glycol (PEG) molecule.

In a third aspect of the present invention, there is provided a method of producing a protein drug conjugate which comprises contacting a protein with a linker capable of providing site specific conjugation via a lysine or cysteine group present on the protein, preferably comprising a nitrogen containing aromatic heterocyclic ring comprising a vinyl tuent, wherein the linker is bound to a drug. In an alternative aspect, there is provided a method of producing a protein drug conjugate which comprises contacting a protein with a linker capable of providing site ic conjugation via a lysine or cysteine group present on the protein, preferably comprising a nitrogen containing aromatic heterocyclic ring comprising a vinyl substituent, and subsequently binding the linker to a drug.

Preferably the method comprises contacting the protein (e.g. the antibody) having at least one reactive thiol group with the linker which comprises a functionalising reagent, comprising a nitrogen ning heterocyclic aromatic ring having a vinyl substituent capable of reacting with at least one thiol group of the protein (e.g. the antibody), wherein the linker onalising reagent is covalently linked to a alkylene glycol) molecule, so that the vinyl substituent of the linker functionalising reagent reacts with the thiol group of the protein (e.g. the dy), thereby to covalently link the linker poly-(alkylene glycol) molecule to the protein (e.g. the antibody).

In addition, the methods of the present invention may involve one or more steps in addition to the step of reacting the functionalising reagent and the protein (e.g. the dy) to link them together. By way of example, the s may include an initial step of reacting a precursor functionalising reagent comprising a nitrogen containing cyclic aromatic ring having a vinyl substituent with the poly-(alkylene glycol) le to produce the functionalising reagent. ln embodiments of the present invention in which the protein (e.g. the antibody) does not include a suitable thiol group, or does not include a suitable thiol group in a desired position in its polypeptide chain, the present invention may comprise the initial step of modifying the protein (e.g. the antibody), e.g. by chemical reaction or site directed mutagenesis, to produce a variant polypeptide having a thiol group at one or more desired positions of the ptide. Preferably, this is done by replacing one or more of the amino acids in the polypeptide chain of the protein (e.g. the antibody) with a ne residue.

Preferably, the reactive thiol group, whether it is t naturally in the protein (e.g. the antibody) or has been introduced, is part of a cysteine amino acid residue.

Additionally, or alternatively, the present invention provides albumin in combination with the , wherein the linker is as described above, in relation to the first embodiment of the present invention.

As will be appreciated, the n ed for use in the present invention is serum albumin, which is a blood plasma protein produced in the liver. ably the albumin is human albumin.

Preferably the linker comprises a poly-(alkylene glycol) molecule, most preferably a polyethylene glycol (PEG) le.

Additionally, or alternatively, the present invention provides a protein drug conjugate comprising albumin, a linker and a drug (e.g. a cytotoxin), wherein the linker and drug (e.g. cytotoxin) are as described above in relation to the protein drug conjugate and/or the ADC invention. In ular, it is preferable that the linker ses a poly-(alkylene glycol) molecule, most preferably a polyethylene glycol (PEG) molecule, which binds to the drug (e.g. the cytotoxin).

The present invention also provides a protein drug conjugate comprising albumin, a linker and a therapeutic e or polypeptide. The linker and therapeutic peptide or polypeptide are as described above, and it is preferable that the linker comprises a poly-(alkylene glycol) molecule, most preferably a polyethylene glycol (PEG) le, which binds to the therapeutic peptide or polypeptide.

Additionally, or alternatively, as described above, the basic poly-(alkylene glycol) structure may be provided with one or more reactive functional groups such as hydroxy, amine, ylic acid, alkyl halide, azide, succinimidyl, or thiol groups to facilitate the reaction of the poly-(alkylene glycol) molecule with the drug, i.e. the xic or therapeutic peptide or polypeptide.

Additionally, the linker poly-(alkylene glycol) structure may also be reacted with a second linker via the linker arm to provide a homobifunctional linker, so that the vinyl group of one linker can be reacted with the thiol group of the drug, i.e. the cytotoxic or therapeutic peptide or polypeptide, and the vinyl group of the other linker can be conjugated to a thiol group of a protein, for example n. In this embodiment the protein drug conjugate, e.g. albumin drug conjugate will comprise two linker molecules, which will facilitate the joining of the albumin to thiol containing cytotoxins, therapeutic peptides or therapeutic polypeptides.

In a r ment of the present invention there is ed a method which comprises contacting albumin with a linker, which comprises a functionalising reagent, comprising a nitrogen containing heterocyclic ic ring having a vinyl substituent capable of reacting with the free thiol group of albumin, wherein the linker functionalising reagent is covalently linked to a poly-(alkylene glycol) molecule, so that the vinyl substituent of the linker onalising reagent reacts with the thiol group of the n, thereby to covalently link the linker poly- (alkylene glycol) molecule to albumin.

This further method may comprise the initial step of modifying the albumin, e.g. by chemical reaction or site directed mutagenesis, to produce a variant having a thiol group at one or more desired positions of the ptide, e.g. to introduce a solvent exposed cysteine residue. Preferably, this is done by replacing one or more of the amino acids in the polypeptide chain of the albumin with a cysteine residue.

In a further aspect, the present invention provides a protein drug conjugate as defined above for use in therapy.

In one embodiment, there is provided an ADC as defined above for use in therapy.

Preferably the ADC is intended for use in anticancer therapy.

In a further embodiment, the t invention provides an albumin drug ate as defined above for use in therapy. Preferably the albumin drug conjugate is intended for use in anticancer, antinociceptive, antidiabetes, antitumor or antiviral therapy.

In a further aspect, the present invention provides a protein drug conjugate formed by the reaction of a globular protein, a linker and a drug, wherein the ar protein is an antibody or fragment thereof comprising at least one reactive thiol group and wherein linker comprises a nitrogen containing heterocyclic aromatic ring comprising a vinyl substituent represented by the ing formulae, 1 N R 2 (III) R1 is selected from; ? (CH2)n - C(O)- R, or ? (CH2)m – S – (CH2)n – C(O) – R, or ? (CH2)n - CH(COR)2, or, ? (CH2)m – S – (CH2)2CH(COR)2, or, wherein, - R2 is selected from the same group of molecules as R1, hydrogen, or an alkyl group, - n is any integer from 0 to 10, - m is any integer between 0 and 10, - R is a hydroxide (OH), amine or a poly-(alkylene glycol) group; wherein the globular protein is conjugated to the linker through the conjugation of the thiol group to the vinyl tuent; and wherein the drug is ated to the linker through the R1 group, the R2 group, or the R1 group and the R2 group.

In a further embodiment, the antibody is a monoclonal antibody.

In a further embodiment, the drug is a cytotoxin or a therapeutic peptide or polypeptide.

In a further embodiment, the xin is a biologically active cytotoxic material.In a further embodiment, the cytotoxin is an anticancer drug.

In a further embodiment, the linker comprises a poly-(alkylene glycol) group.

In a further embodiment, the poly-(alkylene glycol) group is a polyethylene glycol (PEG).

In a further ment, the poly-(alkylene glycol) structure is provided with one or more reactive functional groups including hydroxy, amine, carboxylic acid, alkyl halide, azide, imidyl or thiol groups.

In a further embodiment, the protein drug conjugate further comprises an er linker.

In a further embodiment, the extender linker is enzyme cleavable.

In a further embodiment, there is a method of producing the protein drug conjugate, which comprises contacting the protein with the linker which is bound to the drug.

In a further ment, the protein drug conjugate is produced comprising contacting the protein with the linker, and subsequently contacting the linker with the drug.

In a further embodiment, there is an l step of reacting a precursor linker sing a en containing heterocyclic aromatic ring having a vinyl substituent with a poly-(alkylene glycol) molecule to produce the linker.

In a further embodiment, there is an initial step of modifying the protein to produce a variant polypeptide having a thiol group at one or more desired ons of the polypeptide.

In a further embodiment, there is a drug protein conjugate for use in therapy.

Embodiments of the t invention will now be described by way of example and not limitation with reference to the accompanying figures.

Brief Description of the Figures Figure 1. Relative rates of reaction for example linkers in accordance with the present invention and glutathione.

Figure 2. RP-HPLC analysis of PL13 free acid reactivity with N-acetyl cysteine (NAC) after 24 hours at three different pHs (7.0, 7.5 and 8.0).

Figure 3. MS data to demonstrate formation of PLNAC Adduct at pH 7.0.

Figure 4. MS data to demonstrate formation of PLNAC Adduct at pH 8.0.

Figure 5. RP-HPLC analysis of PL13 free acid reactivity with NAC at pH 8.0.

Figure 6. C analysis of PL13 free acid reactivity with a mixture of amino acids (Tyr/Lys/His/NAC) at pH 7.0.

Figure 7. MS data to demonstrate formation of PL13-NAC in reactivity with amino acid mixture at pH 7.0.

Figure 8. RP-HPLC analysis of PL13 free acid reactivity with 10 equivalents of Lysine at pH 7.0.

Figure 9. RP-HPLC analysis of PL13 free acid reactivity with 10 equivalents of ic acid.

Figure 10. ure of PL13-val-citaminobenzoyl-M MAE.

Figure 11. MS analysis of PL13-val-citaminobenzoyl-MMAE.

Figure 12. HIC and UV-Vis profiles of Trastuzumab-maleimide-val-cit aminobenzoyl-M MAE after 1 hour of reaction.

Figure 13. HIC and UV-Vis profiles of the zumab-PL13-val-cit aminobenzoyl-MMAE ate at 1, 8 and 16 hours of incubation with 1.25 molar excess of drug-linker over thiol.

Figure 14. HIC e of the Trastuzumab-PL13-val-citaminobenzoyl-MMAE conjugate at 16 hours of incubation with 5 molar excess of drug-linker over thiol.

Figure 15. HIC profile of zumab-PL13-val-citaminobenzoyl-MMAE obtained via conjugation of 10 molar excess of PL13-val-citaminobenzoyl- MMAE after 16 hours of incubation.

Figure 16. PLRP profile of Trastuzumab-PL13-val-citaminobenzoyl-MMAE conjugated at 10 molar excess of PL13-val-citaminobenzoyl-MMAE after 16 hours of tion.

Figure 17. PLRP profile of double desalted Trastuzumab-PL13-val-cit aminobenzoyl-MMAE.

Figure 18. HIC analysis of the Trastuzumab-PL13-val-citaminobenzoyl-M MAE conjugate in the presence of NAC at O, 24 and 48 hours.

Figure 19. PLRP profile of the Trastuzumab-PL13-val-citaminobenzoyl-MMAE conjugate in the presence of NAC at O, 24 and 48 hours.

Figure 20. HIC analysis of the Trastuzumab-maleimide-val-citaminobenzoyl- MMAE conjugate in the ce of NAC at O, 24 and 48 hours.

Figure 21. Analysis of Trastuzumab-maleimide-val-citaminobenzoyl-MMAE (A) and Trastuzumab-PL13-val-citaminobenzoyl-MMAE (B) conjugates by SEC chromatography.

Figure 22. Non-reducing SDS-PAGE analysis of Trastuzumab-PL13-val-cit aminobenzoyl-MMAE and Trastuzumab-maleimide-val-citaminobenzoyl- MMAE.

Figure 23. Structure of H-PEG4-OSu linker.

Figure 24. ESl-MS analysis of PL13-NH-PEG4-COOH linker intermediate.

Figure 25. is of Trastuzumab cross-linking via SMCC or H-PEG4- OSu linker by reducing SDS-PAGE.

Figure 26. Structure of PL13-NH-PEG4- val-citaminobenzoyl-M MAE linker.

Figure 27. HIC profile of Trastuzumab-maleimide-val-citaminobenzoyl-MMAE (A) and Trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl-M MAE (B).

Figure 28. PLRP profile of Trastuzumab-maleimide-val-citaminobenzoyl- MMAE (A) and Trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl-MMAE (B).

Figure 29. DAR calculation for Trastuzumab-PL13-NH-PEG4-val-cit—MMAE and Trastuzumab-mal-val-cit-M MAE based on the PLRP elution profile of Figure 28.

Figure 30. SEC is of Trastuzumab-mal-val-cit-MMAE (A) and Trastuzumab-PL13-NH-PEG4-val-cit-MMAE (B).

Figure 31. Representative HIC (A) and PLRP (B) profiles of Trastuzumab-PL13- NH-PEG4-val-citaminobenzoyl-MMAE achieved after process optimisation at 1 mg scale.

Figure 32. HlC profiles of Trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl- MMAE (A) and Trastuzumab-maleimide-val-citaminobenzoyl-MMAE (B) achieved at 150 mg scale.

Figure 33. SEC es of Trastuzumab-PL13-NH-PEG4-val-cit-aminobenzoyl- MMAE (A) and Trastuzumab-maleimide-val-cit-aminobenzoyl-MMAE (B) achieved at 150 mg scale.

Figure 34. HlC profile of Thiomab-PL13-NH-PEG4-val-cit—MMAE at pH 7.0 (solid line) and pH 7.5 (dashed line).

Figure 35. PLRP profile to demonstrate de-drugging for trastuzumab-malemide- val-citaminobenzoyl-MMAE.

Figure 36. PLRP e to demonstrate de-drugging for trastuzumab-PL13-NH- PEG4-val-citaminobenzoyl-M MAE with a DAR of 3.8.

Figure 37. In vitro stability of ADCs: trastuzumab-malemide-val-cit enzoyl-MMAE, Mal-ADC (A) vs trastuzumab-PL13-NH-PEG4-val-cit—4- aminobenzoyl-MMAE, PL13-ADC (B) Figure 38. In vivo stability of ADCs: PL13-ADC vs Mal-ADC Figure 39. Cell kill data for SKBR3 cell line (A), BT474 cell line (B) and JIMT-1 cell line (C).

Figure 40. Multi-dose xenograft data to demonstrate ADC ty.

Figure 41. Multi-dose xenograft data to show ADC effect on tumour growth.

Figure 42. Tumour maturity histopathology data Figure 43. Comparative data for conjugation of 20 KDa PEG-PL12 and PEG- PL13 versus 20 KDa PEG-maleimide to reduced albumin at pH 7.4 Figure 44. Optimisation of PL11 (A), PL12 (B) and PL13 (C) ation to albumin.

Figure 45. Analysis of PL11 (A), PL12 (B) and PL13 (C) conjugation to albumin by ESl-MS.

Figure 46. Stability of Albumin-PL-12 conjugate in the presence of 1 mM GSH demonstrated by ESl-MS.

Figure 47. Structure of PL13-NH-PEG4-val-citaminobenzoyl-Doxorubicin Figure 48. Comparative data for conjugation of H-PEG4-val-cit—4- aminobenzoyl-doxorubicin linker to albumin, thioalbumin (single mutant) and thioalbumin (double mutant).

Figure 49. Non-reducing SDS-PAGE analysis of albumin-PL13-NH-PEG4-val-cit— 4-aminobenzoyl-doxorubicin conjugates. (1) Thioalbumin-double -DOX; (2) bumin-single mutant-DOX; (3) Albumin-DOX; (M)-protein marker.

Figure 50. SEC analysis of albumin-PL13-NH-PEG4-val-citaminobenzoyl- doxorubicin conjugates: Albumin (A) and albumin-DOX ate (B); Thioalbumin (single mutant) (C) and Thioalbumin-DOX (single mutant) (D); Thioalbumin (double mutant) (E) and Thioalbumin-DOX (double ) (F).

The methods of the present invention are capable of providing protein drug conjugates, such as ADCs or albumin drug conjugates, in accordance with the present invention which comprise an antibody or albumin bound to a linker, which in turn is bound to a drug, such as a cytotoxin or a therapeutic peptide or ptide.

In the t invention, nces to dies include immunoglobulins whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antigen binding . Antibody fragments are also contemplated which comprise antigen binding domains including Fab, scFv, Fv, dAb, Fd fragments, diabodies, triabodies or nanobodies.

It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable , or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0 184 187 A, GB 2,188,638 A or EP 0 239 400 A. dies can be modified in a number of ways and the term should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the ed specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding , or equivalent, fused to another polypeptide are therefore ed. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A.

In the prior art it has been shown that fragments of a whole antibody can perform the function of binding antigens. es of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 s; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb nt (Ward, E.S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment sing two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a e linker which allows the two s to associate to form an antigen binding site (Bird et al, Science, 242; 423-426, 1988; Huston et al, PNAS USA, 85: 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/O9965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; Holliger et al, P.N.A.S. USA, 90: 6444-6448, 1993). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al, Nature h, 14: 1239-1245, 1996). Minibodies comprising a ned to a CH3 domain may also be made (Hu et al, Cancer Res, 56: 3055-3061, 1996).

Accordingly, such g fragments are contemplated by the present invention.

In preferred embodiments, the methods disclosed herein employ reagents and conditions that are well adapted for binding a protein, such as an antibody, via a linker to a drug, such as a cytotoxin. In particular, the on conditions that are used in the present method helps to avoid the problems that tend to occur when using prior art reagents such as maleimide, which have a tendency to produce a mixture of different products with a range of different properties. More especially, as can be seen from the experimental data below, methods of producing protein drug conjugates, such as ADCs, which e linkers according to the present invention, avoid the problems associated with maleimide crosslinking.

As mentioned above, the protein of interest (e.g. the antibody) for the protein drug conjugate (e.g. ADC) composition may be bound to the linker using existing thiol groups or by ucing thiol groups in an initial step of the method, for example by reacting one or more functional groups of the protein (e.g. antibody) to produce a thiol group, or by introducing a thiol group or a precursor f into the protein (e.g. antibody). By way of example, this may involve the step of introducing a cysteine residue into the protein (in the example an dy) at a site where it is desired to bind the linker to the protein (e.g. antibody). This may be useful in situations where a convenient ne residue for reaction according to the present invention is not present in a starting or wild-type polypeptide/protein. Conveniently, this may be achieved using site directed mutagenesis of the protein, such as an antibody polypeptide, the use of which is well established in the art.

Alternatively or additionally, an initial reduction step may be required where the protein is commercial grade albumin as the majority of cysteine thiols present are capped, as sed in further details below mental Data and Discussion The following experimental data was mainly produced utilising the linker le identified as, and ed to herewith as, PL13, in its free acid or NH - pegylated form, as shown below. or [13] 1. Demonstration of PL11, PL12 and PL13 linker reactivity with glutathione.

Glutathione contains a thiol group which is readily available for conjugation, and can provide a good model for proteins to assess the suitability of linkers for use in the present invention for utility in protein drug ates, such as ADCs.

Figure 1 shows three linker group examples according to the present invention and demonstrates their ability to conjugate to the thiol groups present in glutathione. The examples in Figure 1 are PL11, PL12 and PL13, and their structures are shown below. 2. Determination of PL13 Free Acid Selectivity The data given below demonstrates that linkers in ance with the present invention show specificity for cysteine groups. PL13 free acid is identified as; l [13] / OH Reactivity of PL13 free acid with N-Acety/ cysteine at pH 7. 0, 7.5 and 8.0 PL13 free acid was reacted with 2 molar equivalents of N-acetyl cysteine (NAC) at three separate pHs: 7.0, 7.5 and 8.0. Reactions were carried out in a methanol/phosphate buffered saline (PBS) solution (ratio of 9:1), buffered to the appropriate pH at room temperature (RT). RP-HPLC analysis with a gradient of 1 % to 50 % B (Acetonitrile with 0.1 % TFA ) over 15 minutes and detection at 254 nm was undertaken to monitor addition of free thiol to the vinyl group of PL13 over time.

Method: 210 uL of 2.61 mM PL13 free acid in methanol was mixed with 22 uL of 50 mM NAC in buffer to give a final tration of 2.37 mM PL13 free acid and 4.74 mM NAC.

Results: The PL13-NAC adduct was eluted at ion time of 5.1 minutes at all pHs after 24 hours (see Figure 2). The amount of product (PL13-NAC adduct) was similar at pH 7.0 and 8.0. The reactivity of PL13 free acid at pH 7.5 was slower. In addition, an unidentified peak was eluted at 4 minutes.

ESI-MS is confirmed the presence of the PL13-NAC adduct at all analysed pHs. MS trace data is ed herewith to show this analysis as performed at pH 7.0 (Figure 3.), and pH 8.0 (Figure 4.). vity of PL13 free acid with N-Acety/ cysteine at pH 8.0 PL13 free acid was d with 2 molar equivalents of NAC in methanol/PBS solution (9:1), pH 8.0. The progress of the reaction was monitored by RP-HPLC using a gradient of 1 % to 50 % B over 15 minutes with detection at 254 nm.

Samples were analysed at 0, 1, 4 and 72 hours of incubation at RT (see Figure Method: 100 pL of 2.61 mM PL13 free acid in methanol was mixed with 11 pL of 50 mM NAC in buffer at pH 8.0 to give a final concentration of 2.35 mM PL13 free acid and 5 mM NAC.

Results: Addition of PL13 free acid to NAC is slow over the first 4 hours. After 72 hours of incubation ~70 % of PL13 free acid was converted to product (PL13-NAC adduct) at pH 8.0.

Reactivity of PL13 free acid with 10 equivalents of Tyrosine, ine, Lysine and N-Acety/ cysteine at pH 7.0 PL13 free acid was challenged with 10 equivalents of each amino acid (tyrosine (Tyr), histidine (His), Lysine (Lys) and N-acetyl cysteine (NAC)) at pH 7.0. The reaction was analysed by RP-HPLC using a gradient of 1 to 5O % B over 15 minutes with detection set at 254 nm.

Method: 50 pL of 2.61 mM PL13 in methanol was mixed with 260 pL of 20 mM s/Lys and NAC in buffer to give a final concentration of 0.42 mM PL13 and 4.2 mM of each of Tyr, His, Lys and NAC.

PL13 free acid reacted selectively with NAC in the presence of Tyr, His and Lys at pH 7.0 (see Figure 6). Figure 7 shows the MS data to trate that only the PL13 NAC adduct was formed.

The addition reaction between PL13 and NAC increased significantly at 10 molar excess of amino acid. Conversion of PL13 free acid to the desired PL13-NAC adduct is 90 % complete after 4 hours at room temperature (RT) and is fully complete in <18 hours.

The results obtained demonstrate the selectivity of the free acid form of the linker molecule in accordance with the present ion for cysteine reactivity via the vinyl group of the linker molecule.

RP—HPLC analysis of PL13 free acid reactivity with lysine.

PL13 free acid was challenged with 10 equivalents of Lys in PBS buffer, pH 7.4 at RT. The reaction was analysed by RP-HPLC using a gradient of 12 to 5O % acetonitrile in water over 30 s with detection set at 270 nm. uL of 4 mM PL13 in methanol was mixed with 50 uL of 8 mM Lys solution and 40 uL of PBS buffer pH 7.4 to give a final concentration of 0.4 mM PL13 and 4.0 mM Lys. The on was analysed by RP-HPLC using a gradient of 12 to 5O % acetonitrile in water over 30 minutes with detection set at 270 nm.

Results: PL13 free acid did not react with Lys at pH 7.4 as no peak is observed in the RP- HPLC analysis corresponding to a PL13-Lys adduct (see Figure 8).

The results ed demonstrate that no adduct is formed by the incubation of PL13 free acid with Lys. This data supports the cysteine group selectivity of the free acid form of linkers of the present invention. It should be appreciated that no such cysteine icity is exhibited by maleimide and as such the linkers of the present invention present a benefit in this regard.

While the above data shows the cysteine specificity ted by the linkers of the present invention, it is readily appreciated that lysine specificity may alternatively be achieved if the linker is modified by methods well known in the art.

Reactivity of PL13 free acid with Aspartic acid PL13 free acid was treated with 10 equivalents of aspartic acid (Asp) at pH 7.0 and at RT. Analysis was performed by RP-HPLC at gradient of 1 % to 5O % B over 15 s with detection at 254 nm.

Method: 50 uL of 2.61 mM PL13 free acid in methanol was mixed with 280 uL of 50 mM Asp in buffer to give a final concentration of 0.42 mM PL13 free acid and 4.2 mM Asp.

Results: PL13 free acid is stable in the presence of Asp at pH 7.0 over 18 hours at RT (see Figure 9).

MS spectra substantiates that no adduct is formed by the incubation of PL13 free acid with Asp (data not shown). Again, this data supports the cysteine group selectivity of the linkers of the present invention. It should be appreciated that no such cysteine specificity is exhibited by ide and as such the s of the present invention present a benefit over this. 3. Synthesis of PL13-VaI-CitaminobenzoyI-MMAE Cytotoxic Drug Linker al-citaminobenzoyl-MMAE was synthesised by a nt approach, which will be familiar to the person skilled in the art.

Method: PL13 free acid was coupled to the free amino terminal of H-val-cit aminobenzoyl-M MAE, an exemplary cytotoxin, via a HOBt active ester method.

Results: 3.1 mg of al-citaminobenzoyl-MMAE (see Figure 10) was synthesised.

The material was solubilised in dimethyl-acetamide (DMA) to afford a drug linker solution at a concentration of 50 mM.

RP-HPLC confirmed purity of the al-citaminobenzoyl-MMAE linker (data not shown).

ESl-MS analysis med the identity of the cytotoxic drug linker (expected MW is 1296.67 Da, experimental result gave MW of 1296.5 Da) (see Figure 11). 4. Generation of Trastuzumab-PL13-Val-Citaminobenzoyl-MMAE and Trastuzumab-maleimide-val-citaminobenzoyl-MMAE 20 mg of Trastuzumab-PL13-val-citaminobenzoyl-MMAE ate, an exemplary ADC of the present invention, and 20 mg of Trastuzumab-maleimide- val-citaminobenzoyl-M MAE conjugate, an ADC comprising a known maleimide linker, were produced. ation of PL13-val-citaminobenzoyI-MMAE and maleimide-vaI-cit aminobenzoyI-MMAE to Trastuzumab Method: Trastuzumab was reduced to allow conjugation of 3-4 drugs per trastuzumab molecule. A detailed method for the reduction of trastuzumab is not provided as this should be well known by the person skilled in the art.

Maleimide-val-citaminobenzoyl-MMAE was ated to Trastuzumab at 1.25 molar excess over free thiol at pH 7.0. The reaction was performed for 1 hour at RT. The conjugation reaction was quenched by an excess of NAC.

Analysis of the Trastuzumab conjugate was accomplished by Hydrophobic Interaction Chromatography (HIC) using a Tosoh butyl-NPR (4.6x3.5, 2.5pm) column and by UV-VIS spectroscopy. MMAE has a distinctive UV absorbance at 248 nm (8248: 1500 M'1cm, 8280: 15900 M'1cm).

PL13-val-citaminobenzoyl-MMAE was coupled to Trastuzumab at 1.25, 2.5, 5 and 10 molar excess over free thiol. Reactions were carried out for 16 hours at pH 7.0. ation reactions were analysed by both HIC (separation on a Tosoh NPR column) and PLRP chromatography (separation on a PLRP column-2.1mmx5cm, 5pm). Trastuzumab conjugates were examined by UV-VIS spectroscopy. Both the MMAE cytotoxic drug and PL13 linker contribute to UV absorbance at 248 nm.

Results: Reaction of maleimide-val-citaminobenzoyl-MMAE with Trastuzumab was completed within 1 hour using a ratio of 1.25 drug over free thiol (see Figure 12).

The numbers in Figure 12 designate the amount of drug conjugated to a full length antibody. The inlet is the UV-Vis profile which represents an increase of absorbance at 248 nm due to conjugation of maleimide-val-citaminobenzoyl- MMAE to Trastuzumab. 10 mg of the zumab-maleimide-val-cit aminobenzoyl-MMAE conjugate (Drug to Antibody Ratio (DAR) 2.2) was obtained and set aside for in vitro studies, as discussed further below.

The rate and efficiency of the PL13-val-citaminobenzoyl-MMAE reaction is much slower than the maleimide-val-citaminobenzoyl-MMAE due to low solubility of the linker (see Figure 13). Conjugation was performed at 1.25 molar excess of drug-linker over thiol. The inlet is the UV-Vis profile which depicts an increase of absorbance at 248 nm due to coupling of al-cit aminobenzoyl-MMAE to Trastuzumab within 16 hours. A slow se in the rate of PL13-val-citaminobenzoyl-MMAE conjugation to Trastuzumab was ed by applying 5 molar excess of drug-linker over thiol group (see Figure 14). In Figure 14, the numbers designate the amount of drug conjugated to a full length antibody. DL indicates unconjugated drug.

Conjugation of PL13-val-citaminobenzoyl-MMAE to Trastuzumab at 10 molar excess and in the presence of 5O % propylene glycol resulted in 90 % yield within 4 hours (see Figure 15 and Figure 16). In Figure 15, the numbers designate the amount of drug conjugated to a full length antibody. DL indicates ugated drug. In Figure 16, the numbers designate the amount of drug ated to light (L) or heavy (H) chain of the dy. 7.2 mg of Trastuzumab-PL13-val-cit enzoyl-MMAE ate (DAR 3.03), was obtained and set aside for in vitro studies, as discussed further below.

Determination of Drug Antibody Ratio (DAR) for Trastuzumab-PL13-val-cit aminobenzoyl—MMAE and Trastuzumab-ma/eimide-val-citaminobenzoyl- MMAE.

HIC and PLRP chromatography are often applied to characterize average drug- load and drug-load distribution for cysteine-linked ADCs. Determination of e drug-load and drug-load distribution is a crucial ute as it effects the potency and pharmacokinetics of the ADC.

Results: HIC characterisation of Trastuzumab-maleimide-val-citaminobenzoyl-M MAE resulted in a DAR calculation of 2.2 with 1.8 % unconjugated Trastuzumab (see Figure 12). The DAR calculation from the HIC profile is determined by well known methods in the art, as discussed below in relation to Figure 27.

HIC analysis of Trastuzumab-PL13-val-citaminobenzoyl-MMAE resulted in peaks which did not resolve well enough to support DAR determination (see Figure 15). However, this data is included here for completeness. HIC was used to calculate the amount of Trastuzumab with DAR 0 calculated to be ~8.4 %.

Separation of dithiothreitol (DTT) reduced Trastuzumab-PL13-val-cit aminobenzoyl-MMAE via a PLRP column afforded well resolved peaks (Figure 16) ponding to unconjugated or drug conjugated antibody light and heavy chains. A DAR of 3.0 was calculated for Trastuzumab-PL13-val-cit aminobenzoyl-MMAE (see Figure 17). The table in Figure 17 depicts the DAR calculation for Trastuzumab-PL13-val-citaminobenzoyl-MMAE based on the percentage area of unconjugated and drug loaded light and heavy .

. Stability s of Trastuzumab-PL13-Val-Citaminobenzoyl- MMAE and Trastuzumab-maleimide-Val-Citaminobenzoyl-MMAE Stability of drug-linker for the trastuzumab-maleimide-val-citaminobenzoyl- MMAE and trastuzumab-PL13-val-citaminobenzoyl-MMAE ates was evaluated in the presence of NAC in PBS .

Method: Each ADC (at concentration of 1-2 mg/mL), was incubated with 1 mM NAC in PBS buffer for 24 hours at 37°C.

Results: The trastuzumab-PL13-val-citaminobenzoyl-MMAE conjugate was not influenced by the presence of NAC in buffer (see s 18 and 19).

Trastuzumab-maleimide-val-citaminobenzoyl-MMAE showed decreased stability in the presence of NAC (see Figure 20). In Figure 18, the numbers designate the amount of drug conjugated to a full length antibody. DL indicates unconjugated drug.

Figure 19 in particular demonstrates there is no profile change for the PL13 containing ADC resulting from challenge from free thiol, indicating stability of the construct. 6. SDS-PAGE and SEC Analysis of Trastuzumab-PL13-Val-Cit aminobenzoyl-MMAE and Trastuzumab-maleimide-Val-Cit aminobenzoyl-MMAE Trastuzumab-maleimide-val-citaminobenzoyl-MMAE and zumab-PL13- taminobenzoyl-M MAE were evaluated by SEC chromatography and non- reducing SDS-PAGE.

Results: Trastuzumab-maleimide-val-citaminobenzoyl-MMAE is present as 99.5 % monomer (see Figure 21 (A)). Trastuzumab-PL13-val-citaminobenzoyl-MMAE is present as 93.5 % monomer (see Figure 21 (8)).

SDS-PAGE is under non-reducing conditions showed the presence of le bands in both the trastuzumab-PL13-val-citaminobenzoyl-MMAE and Trastuzumab-maleimide-val-citaminobenzoyl-MMAE samples due to the disruption of inter-chain disulphide bonds in the antibody during alkylation of cysteine residues with linker-drug moieties (see Figure 22). In Figure 22, lane 1 represents the analysis of a 5 uL of sample of trastuzumab-PL13-val-cit enzoyl-M MAE loaded on the gel at a concentration of 1.8 mg/mL and lane 2 represents the analysis of a 5 uL of sample of trastuzumab-maleimide-val-cit aminobenzoyl-MMAE loaded on the gel at a concentration of 5 mg/mL. The ity of full length antibody is still sustained due to strong non-covalent interactions between heavy and light chains which was confirmed by SEC analysis. 7. Synthesis of PL13-NH-PEG4-OSu Heterobifunctional Linker The hydrophilic PEG (PEG4) was uced to the linker molecule linker arm portion, to provide PL13-NH-PEG4-OSu, to improve linker solubility over the example given under section 1 above (see Figure 23).

Method: PL13 free acid (as described above) was coupled to 1-Amino-3, 6, 9, 12- tetraoxapentadecanoic acid. Activation of the carboxyl group to the desired succinimidyl ester was undertaken using N,N-dicyclohexylcarbodiimide (DCC) 2.5 eq./HOSu coupling in anhydrous dichloromethane (DCM). Unreacted DCC and a diisopropylurea by-product were removed via filtration from cold DCM.

Results: ESl-MS of PL13-NH-PEG4-COOH (see Figure 24), confirmed the linker identity. ison of cross-reactivity of PL13-NH-PEG4-OSu and SMCC linkers The extent of Trastuzumab cross-linking via imidyl trans (maleimidylmethyl)cyclohexanecarboxylate (SMCC), a known linker, and PL13-NH-PEG4-OSu was evaluated by ng SDS-PAGE.

Method: Trastuzumab at a final concentration of 2 mg/mL was incubated with 10 fold excess of SMCC or PL13-NH-PEG4-OSu. Samples were incubated at RT.

Results: It is evident from the SDS-PAGE that PL13-NH-PEG4-OSu shows less high molecular weight bands than the SMCC linker (see Figure 25, lanes 5-9). There are some higher molecular weight bands on the H-PEG4-OSu SDS- PAGE but these are present to some extent in the starting Trastuzumab lane as well (see Figure 25, lane 5).

In contrast to the linkers of the present invention, the SMCC linker induces trastuzumab cross-linking due to non specific reactivity of the maleimide group particularly towards amine side chains of lysine residues. 8. Synthesis of PL13-NH-PEG4-val-citaminobenzoyl-MMAE To improve further the lity of the previously synthesised PL13-val-cit enzoyl-MMAE cytotoxic drug linker, a short hydrophilic PEG unit was introduced to the molecule (see Figure 26). The PEG unit also introduces a ‘spacer’ to the ADC linker arm, increasing the separation of the MMAE cytotoxic payload and the antibody attachment point through PL13 by some 39 atoms (versus 22 atoms previously in PL13-chMAE). It is believed this r" should increase the rotational flexibility of the PL13 unit, which may influence the cs of the Michael addition to free thiol.

Method: 40 mg of crude Fmoc-val-citaminobenzoyl-MMAE was purified. The N-Fmoc group was then removed by ysis and the amine functionalised cytotoxic nd was purified to afford 24 mg of material. This was used to couple PL13-NH-PEG4-COOH via standard HOBt active ester chemistry.

Results: PL13-NH-PEG4-val-citaminobenzoyl-MMAE was synthesised. This molecule represents an example of a cleavable ADC system in accordance with the present invention. 9. Conjugation of PL13-NH-PEG4-val-citaminobenzoyl-MMAE and Maleimide-val-citaminobenzoyl-MMAE to Trastuzumab.

: Trastuzumab was reduced with 2.4 times excess of tris(2-carboxythyl)phosphine (TCEP) for 1 hour at RT in the presence of ethylenediaminetetraacetic acid (EDTA). The antibody was fered into PBS. Trastuzumab, at a concentration of 20 mg/mL, was conjugated to 20 fold excess of PL13-NH-PEG4-val-cit—4- aminobenzoyl-MMAE in the presence of 5 % v/v DMA at RT for 16 hours.

Additionally, maleimide-val-citaminobenzoyl-MMAE linker was coupled to Trastuzumab (20 mg/mL) at 6 molar excess at RT for 1 hour. The ADCs obtained were ed by HIC, PLRP and SEC chromatography.

Results: HIC characterisation of trastuzumab-maleimide-val-citaminobenzoyl-M MAE resulted in a DAR calculation of 4.29 (see Figure 27 A).

HIC characterisation of trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl- MMAE resulted in a DAR calculation of 3.15 (see Figure 27 B).

In Figures 27A and 27B, the s designate the amount of drug conjugated to a full length antibody. The table represents the relative percentage of each peak area from the HIC elution profile. The average DAR is calculated by multiplying the percentage peak area by the corresponding drug load to obtain a weighted peak area. The weighted peak areas are summed and divided by 100 to give a final DAR value.

DAR analysis of DTT d trastuzumab-maleimide-NH-PEG4-val-cit aminobenzoyl-MMAE by PLRP confirmed the average drug load to be 4.3 (see Figure 28 A and Figure 29).

Analysis of DTT reduced trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl- MMAE by PLRP confirmed an average drug load to be 3.8 (see Figure 28 B and Figure 29).

In Figures 28A and 28B, the numbers designate the amount of drug conjugated to light (L) or heavy (H) chain of the antibody.

Analysis of both trastuzumab-maleimide-val-citaminobenzoyl-MMAE (HER- MAL-MMEA) and zumab-PL13-NH-PEG4-val-citaminobenzoyl-MMAE (HER-PLMMEA) on SEC chromatography showed that both samples are represented as monomeric s (see Figure 30 A and B). The table below Figures 30A and 30B shows the ation of high molecular weight species (HMW), monomeric and low molecular weight species (LMVV).

. Optimised conditions for the ation of PL13-NH-PEG4-val-cit aminobenzoyl-MMAE Method: Trastuzumab (23.5 mg/mL) was reduced with 2.4 equivalents of TCEP for 2 hours at RT to yield an average of 4.5 free thiols. The reduced Trastuzumab was conjugated to 20 fold excess of PL13-NH-PEG4-val-cit—MMAE in the presence of 1O % v/v DMA at 30 °C for 18 hours. The conjugate was analysed by HIC and PLRP chromatography (see Figure 31 A and B).

The HIC and PLRP data show a substantial improvement in conjugation efficiency. The higher level of conjugation efficiency has also resulted in a significant reduction in the level of ‘odd’ DAR species and a reduction in the level of ugated antibody. 11. Conjugation of zumab-PL13-NH-PEG4-val-cit-aminobenzoyl- MMAE (A) and zumab-maleimide-val-cit-aminobenzoyl-MMAE (B) at 150 mg scale, demonstrating scalability of the process Method: Trastuzumab (24.53 mg/mL) was reduced with 2.1 equivalents of TCEP for 2 hours at RT. The reduced Trastuzumab was conjugated to 20 fold excess of H-PEG4-val-cit—MMAE in the presence of 1O % v/v DMA at 30 °C for 18 hours.

Trastuzumab at concentration of 25.68 mg/ml was reduced with 1.95 equivalents of TCEP for 2 hours at RT. The reduced Trastuzumab was conjugated to 6 fold excess of maleimide-val-cit-MMAE in the presence of 1O % v/v DMA at RT for 1 The conjugates were analysed by HIC and SEC chromatography (see Figures 32 and 33).

Results: The HIC es showed similar conjugation ency for Trastuzumab-PL13- NH-PEG4-vaI-cit-aminobenzoyI-MMAE (Figure 32A) and Trastuzumab- ma|eimide-vaI-cit-aminobenzoyI-MMAE (Figure 328). SEC (Figures 33A and 338) confirmed that both conjugates are monomers with only a small amount of high molecular weight species present (1.4 - 2.4 0/0). This demonstrates the scalability of the conjugation process developed using the linkers of the present invention. 12. Conjugation of PL13-NH-PEG4-val-cit-aminobenzoyl-MMAE to b, trating successful conjugation to engineered Trastuzumab (Thiomab) Trastuzumab with a cysteine mutation (V205C) introduced in the antibody light chain was conjugated at concentration of 10 mg/ml to 40 fold excess of PL13- NH-PEG4-val-cit—MMAE in the presence of 10 % DMA in buffer pH 7.4.

Conjugation reactions were incubated at 35 °C for 48 hours.

Results: The HIC profile of Trastuzumab (V2015C)-PL13-NH-PEG4-val-cit—MMAE demonstrates that PL13 can be successfully conjugated to antibodies with engineered Cys residues at both pHs (Figure 34). The rate and extent of the conjugation reaction appears to be influenced by the thio| nvironment. 13. Utility of ADCs according to the present invention as ADCs.

ADC conjugation products, as described above in section 8, were further subjected to in vivo and in vitro testing to demonstrate their utility as commercial ADCs.

In vitro stability of trastuzumab-malemide-val-citaminobenzoyl-MMAE and trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl-MMAE in the presence of Method: Stability of drug-linker for the trastuzumab-malemide-val—cit—4-aminobenzoyl- MMAE and trastuzumab-PL13-NH-PEG4-val—cit—4-aminobenzoyI-MMAE conjugates was evaluated in the presence of NAC in PBS buffer.

Results: The trastuzumab-PL13-NH-PEG4-val—cit—4-aminobenzoyI-MMAE conjugate was not influenced by the presence of NAC in the buffer (Figure 36). Trastuzumab- de-val-citaminobenzoyI-MMAE showed decreased stability in the presence of NAC (see Figure 35). Figure 36 in particular demonstrates there is no profile change for the PL13 containing ADC resulting from challenge from free thiol, indicating stability of the uct.

In vitro stability of trastuzumab-malemide-val-citaminobenzoyl-MMAE and trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl-MMAE in mouse plasma The in vitro serum stability of the ADCs Trastuzumab-PL13-NH-PEG4-val—cit— aminobenzoyI-MMAE and Trastuzumab-maleimide-val-cit—aminobenzoyI-MMAE was compared in mouse plasma.

: Trastuzumab-PL13-NH-PEG4-val-cit-aminobenzoyI-MMAE and Trastuzumab- maleimide-val-cit-aminobenzoyI-M MAE were separately spiked into filtered athymic nude mouse plasma to a tration of 0.2 mg/mL. The solutions were mixed and triplicate aliquots of 50 uL were taken and snap frozen in liquid nitrogen (time point — Oh). The plasma solutions of the ADCs were incubated at 37°C for 7 days. Aliquots of 50 uL were pulled in triplicate for each time point: 1, 2, 3, 4, 5, 6 and 7 days, and stored at -80°C until analysis.

Results: ADC stability was monitored by /MS analysis. Plasma samples were pre- purified and trypsin/CNBr digested before MS analysis. Four non-conjugated peptides (two from heavy chain and two from light chain) and two conjugated peptides (one from heavy chain and one from light chain) were selected to monitor the stability of the ADCs. Averaged data from non-conjugated peptides correspond to the stability of the antibody component in mouse plasma (Total- Ab) and data from conjugated peptides show the stability of the ADC conjugate (LC ated, HC conjugated peptide).

This in vitro data revealed that Trastuzumab-maleimide-vaI-cit-aminobenzoyl- MMAE undergoes de-drugging at both the light chain and heavy chain, whereas the antibody component is stable (Figure 37A). Loss of drug from the heavy chain peptide was more rapid when compared to the light chain e, indicating that the rate of drug loss is affected by the ation site.

The zumab-PL13-NH-PEG4-va|-cit-aminobenzoyI-MMAE conjugate retained MMAE drug throughout the 7 day incubation period, showing that PL13 confers stability to both conjugation sites (Figure 378). The observed variability for both conjugates is due to efficiency of sample recovery for the ESI-MS method applied for sample analysis.

In vivo stability of trastuzumab-ma/emide-val-citaminobenzoyl-MMAE and trastuzumab-PL13-NH-PEG4-val-citaminobenzoyI-MMAE in mice Method: In vivo stability was evaluated in mice injected with 5 mg/kg of the zumab- de-vaI-citaminobenzoyI-MMAE and trastuzumab-PL13-NH-PEG4-val- aminobenzoyI-MMAE conjugates. Plasma samples were pre-purified and trypsin/CNBr digested before LC-MS analysis. Two conjugated peptides (one from heavy chain and one from light chain) were selected to monitor the stability of the ADCs.

Results: The stability of conjugated peptides is a result of two events; 1) catabolic degradation of ADCs and 2) loss of drug due to linker instability. Conjugated peptides derived from zumab-PL13-NH-PEG4-val-citaminobenzoyl- MMAE show better stability at 1 day in mouse plasma than peptides derived from zumab-malemide-val-citaminobenzoyl-MMAE. The observed ence is likely due to more rapid drug loss from conjugated peptides derived from trastuzumab-malemide-val-citaminobenzoyl-MMAE than from trastuzumab- PL13-NH-PEG4-val-citaminobenzoyl-MMAE which is consistent with the in vitro mouse plasma data, showing rapid de-drugging for trastuzumab-malemide- val-citaminobenzoyl-MMAE within the first four days of tion (Figure 38); approximately 70% of the drug attached to the light chain and 90% of the drug on the heavy chain was lost within the first two days. In contrast, trastuzumab- PL13-NH-PEG4-val-citaminobenzoyl-MMAE retains more drug due to the linker stability over a longer period of time.

In vitro stability of trastuzumab-ma/emide-val-citaminobenzoyl—MMAE and zumab-PL13-NH-PEG4-val-citaminobenzoyl-MMAE in various cell lines In Figures 39A to 39C SKBRB, BT74? and JIMT-1 cell kill data are shown for the ADCs described above. The SKBRB, BT74? and JIMT-1 cell lines were selected in this test, as they are known to express the Her2 receptor. Additionally, the JIMT-1 cell line is known to be resistant to trastuzumab. ln Figures 39A to 39C "ADC mal" relates to trastuzumab-maleimide-val-citaminobenzoyl-MMAE, and "ADC-PL13" relates to trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl- MMAE. Relative in vitro activities of tested conjugates against three cell lines indicate the data for the trastuzumab-PL13-NH-PEG4-val-citaminobenzoyl- MMAE are able to the trastuzumab-maleimide-val-citaminobenzoyl- MMAE. Accordingly, this data shows that the ADC of the present invention is active and so the presence of PL13 does not have a negative effect on cell killing.

In vivo stability of zumab-ma/emide-val-citaminobenzoyl-MMAE and trastuzumab-PL13-NH-PEG4-val-citaminobenzoyI-MMAE in mice exhibiting breast cancer cell tumours Xenograft data is shown in Figures 40, 41 and 42 in relation to the effect of the ADCs on mice exhibiting breast cancer cell tumours. The breast cancer cell line utilised was BT474 (Her2 expresser, with high susceptibility to trastuzumab).

Mouse weight is used (as shown in Figure 40) to measure the toxicity of the ADC in vivo. It can be seen from the data obtained that the trastuzumab-maleimide- val-citaminobenzoyI-M MAE (ADC-mal) e 40A) and the trastuzumab- PL13-NH-PEG4-val-citaminobenzoyI-MMAE (ADC-PL13) (Figure 408) have a negligible effect on mouse weight over the course of the therapy period. This is indicative of the ADCs of the present invention being of suitable toxicity levels for use commercially.

Figure 41 shows the iveness of the tested ADCs at three doses (0.1, 5 and mg/kg) on tumour growth over a 21 day therapy period. Tumour growth was ined by changes in tumour volumes. Mice treated with both compounds at 0.1 mg/kg enced tumour growth delay relative to the untreated group. In st, all of the animals dosed with the trastuzumab-maleimide-val-cit aminobenzoyI-MMAE (ADC-mal) and the trastuzumab-PL13-NH-PEG4-val—cit—4- enzoyI-MMAE (ADC-PL13) at 5 and 10 mg/kg showed complete responses over the therapy period.

Figure 42 shows the effectiveness of the tested ADCs at three doses (0.1, 5 and mg/kg) on tumour maturity histological phenotype over a 10 day therapy period. Tumour tissues were examined microscopically after oxylin and eosin staining and graded as follows: 0: no xenograft mass; occasionally, isolated tumour cells in sub-cutis or fat tissue 1: small fragmented mass; incomplete epithelial development sequence 2: xenograft showing marked cell loss 3: zonal cell loss with areas of mature tumour 4: intact xenograft mass showing full epithelial development sequence and associated ogy No reduction in the tumour maturity was observed for either trastuzumab-PL13- NH-PEG4-val-citaminobenzoyl-MMAE (ADC-PL13) or trastuzumab- maleimide-val-citaminobenzoyl-MMAE (ADC-mal) dosed at 0.1 mg/kg, with no significant differences observed between the two ADCs. However, zumab- PL13-NH-PEG4-val-citaminobenzoyl-MMAE dosed at 5 and 10 mg/kg revealed significantly reduced tumour maturity starting at days 3 (grade 3 and grade 3/2, respectively). This effect was more pronounced at day 10 (grade 1 for mg/kg and grade 1/0 for 10 mg/kg) with some samples showing no tumour mass. The trastuzumab-maleimide-val-citaminobenzoyl-M MAE dosed at 5 and mg/kg reduced tumour maturity (grade 4/3 and grade 4/3/2, respectively) starting at 3 days, but the s were less pronounced in comparison with animals dosed at corresponding doses of trastuzumab-PL13-NH-PEG4-val-cit aminobenzoyl-MMAE at 3 days. Furthermore, there was a ly greater tumour heterogeneity for ADC-mal than ed for 13 for 5 and 10 mg/kg doses at 10 days. 14. Demonstration 0f PL11-PEG20KDa, PL12-PEG20KDa and PL13-PEG20KDa linker reactivity with albumin As indicated above, the majority of the cysteine thiols t in commercial grade albumin are capped, and so a reduction step is necessary prior to conjugation with linker molecules.

Method: Albumin (221 uM) was reduced with dithiothreitol (DTT) (1 mM) in phosphate- buffered saline (PBS) pH 7.0 containing ethylenediaminetetraacetic acid (EDTA) (1 mM) for 1 hour at room temperature (RT), ed by desalting on a Nap-5 column and quantification by UV measurement. Conjugation of 20 KDa PEG-PL- 12 (1O molar excess) or 20 KDa PEG-PL-13 (1O molar excess) to reduced albumin (20 uM) was performed in PBS, pH 7.4, 1 mM EDTA for 1 day at RT.

Similarly, conjugation of 20 KDa PEG-maleimide (1.1 molar excess) to reduced albumin (20 uM) was performed in PBS, pH 7.4 containing 1 mM EDTA for1 day at RT. The reactions were analysed on SDS-PAGE gel and quantification performed by analysis of protein band density on SDS-PAGE gel using lmageLab re (Biorad).

Results: Only moderate conjugation yields were ed with reduced commercial grade albumin after 1 day. The conjugation of PL13 proceeded in a comparable yield to the maleimide control, whereas PL12 gave a slightly lower yield over the same time period. This is shown in Figure 43.

. Optimisation of PL11, PL12 and PL13 conjugation to n The conjugation of the linker to albumin was determined at pH 5.5 for PL-11 and at pH 5.5, 6.5, 7.5, 8.0 and 8.5 for PL-12 and PL -13.

Method: Conjugation of PL-11 (1O molar excess) to albumin (50 uM) was performed in 50 mM acetate-Na pH 5.5 for 24 hours at 37 °C. Conjugation of PL-12 and PL-13 (1O molar excess) to albumin (50 uM) was performed in the following buffers: 50 mM acetate-Na pH 5.5; PBS 1mM EDTA pH 6.5; PBS 1mM EDTA pH 7.5; 100 mM phosphate; 1mM EDTA pH 8.0; and 100 mM carbonate-Na, 1mM EDTA pH 8.5; for 24 hours at 37 °C.

Upon completion, all albumin samples were desalted on a Nap-5 column into PBS buffer (pH 7.4). The conjugation efficiency was quantified by Ellman’s assay. Ellman’s assay detects and quantifies free ne residues by reacting of 5,5'-Dithio-bis-(2-nitrobenzoic acid) with free thiol groups. The conjugation of linker molecules to the free thiol group of 34Cys in albumin blocks the detection of these groups by Ellman’s t in comparison to the unconjugated n l. A se in the concentration of detectable free thiols was used to quantify amount of linker molecule conjugated to the albumin.

Results: Conjugation of PL-11 to albumin at pH 5.5 proceeded in an 82 % yield. The optimum pH for conjugation of PL-12 (91 %) and PL-13 (94 %) was ined to be pH 7.5. These results are shown in Figure 44. 16. Demonstration of linker-albumin conjugate stability by ESl-MS In addition, n-PL11, Albumin-PL12 and Albumin-PL13 conjugates were analysed by ESl-MS.

Method: Conjugation of PL-11 (2O molar excess) to albumin (50 uM) was performed in PBS containing EDTA (1 mM) at pH 6.5 for 24 hours at 37 °C. Conjugation of PL-12 and PL-13 (1O molar excess) to albumin (50 uM) was performed in PBS containing EDTA (1 mM) at pH 7.5 for 24 hours at 37 °C. The albumin-linker conjugates were analysed by .

Results: Figure 45 shows the ESl-MS results obtained for the albumin-PL-11 conjugation (A), albumin-PL-12 conjugation (B) and the n-PL-13 conjugation (C).

The following albumin species were detected and quantified based on MS signal intensity (note that MW stands for molecular weight): Albumin-PL11 - Unconjugated albumin — 6 % Expected MW of unconjugated Albumin — 66440 (Detected MW = 66445 Da) - n-PL11 conjugate — 90 % Expected MW of albumin-PL11 — 66649 Da (Detected MW = 66650 Da) - Albumin conjugated to two PL11 ~ 4 % ed MW of albuminPL11 — 66858 Da (Detected MW = 66858 Da) Albumin-PL12 - Unconjugated Albumin — 1 % Expected MW of unconjugated Albumin — 66440 (Detected MW = 66441 Da) - Albumin-PL12 — 92 % Expected MW of albumin-PL12— 66663 Da (Detected MW = 66664 Da) - Albumin ated to two PL12 - 7 % Expected MW of albuminPL12 — 66886 Da (Detected MW = 66886 Da) Albumin-PL13 - Unconjugated Albumin — 5 % Expected MW of unconjugated Albumin — 66440 (Detected MW = 66441 Da) - Albumin-PL13 — 94 % Expected MW of albumin-PL13 — 66631 Da (Detected MW = 66632Da) - Albumin conjugated to two PL13 — 1% Expected MW of albuminPL13 - 66822 Da (Detected MW = D66822a) In particular, with nce to Figure 45, peaks (4501), (4504) and (4507) pond to unreacted and oxidised albumin, peaks (4502), (4505) and (4508) correspond to single conjugate albumin and peaks (4503), (4506) and (4509) correspond to double conjugate albumin. 17.Demonstration of stability of albumin-linker conjugates in glutathione The stability of albumin-linker molecule conjugates was determined in the presence of excess hione and ed by ESl-MS over a period of 7 days.

Method: Samples of the albumin-PL-12 conjugate (0.5 mg/mL) were incubated with d glutathione (1 mM) in PBS (pH 7.4) at 37°C for 7 days. The samples were ed on ESl-MS.

Results: Figure 46 shows the results obtained for the albumin-PL-12 conjugate as a representative example. A similar trend was observed for the albumin-PL-11 and albumin-PL-13 conjugates.

Figure 46 shows the ESl-MS results at day 0, 1, 4 and 7. In Figure 46, peaks (4601), (4603), (4605) and (4608) correspond to n, peaks (4602), (4604), (4606) and (4609) correspond to albumin-PL12 ate and peaks (4607) and (4610) correspond to GSH-albumin. ingly, it is shown that: - The major species present is the n-PL12 conjugate (MW = ~66664 - Upon incubation with 1 mM glutathione, there is a very slow increase in Albumin-GSH signal (~8 %, MW = 66752 Da) starting at 4 day of incubation with glutathione and reaching ~13 % on 7th day of incubation.

It can be concluded that albumin-linker conjugates of the present invention demonstrate good stability in a competitive environment. eration of n-doxorubicin conjugates using linkers according to the present invention Method: Albumin, Thioalbumin e cysteine mutant) and Thioalbumin (double cysteine mutant) were conjugated to 14, 20 and 30 fold excess of PL13-NH-PEG4-val-cit- 4-aminobenzoyl-doxorubicin linker e 47), respectively. Reactions were carried out in 147 mM sodium phosphate pH 7.5, in the presence of 10 % v/v DMA and 0.6 mM EDTA, at 37 °C for 2 days in the dark. After this time, the reactions were desalted on PD-10 columns equilibrated in PBS buffer pH 7.4.

The conjugation efficacy was determined by UV-Vis spectroscopy using the doxorubicin absorbance and extinction coefficient at 495 nm (8:8030 M'1, cm'1) and that of albumin at 280 (8:34445 M4, cm'1), with correction for doxorubicin absorbance at 280 nm according to the equation: (AszBOnm—(0.724XAbs495nm)) Albumin concentration = 8280 nm The extent of n-doxorubicin ate aggregation was determined by analysis on non-reducing SDS-PAGE and SEC chromatography. pl of each albumin-doxorubicin conjugate in SDS-PAGE loading buffer was loaded on NuPAGE 4-12% Bis-Tris gel and run for 45 minutes at 200 V. The native fluorescence properties of doxorubicin were used to visualise albumin- doxorubicin conjugates before staining of a gel with Coomassie dye for n species. 5 pl of each conjugate was loaded on to a SEC HPLC column equilibrated with 150 mM sodium phosphate buffer at pH 7.0 and eluted at flow rate of 1 mL/min with detection at 280 nm.

Results: Conjugation of doxorubicin to wild-type albumin and albumin mutants resulted in yields of 55 % for n, 32 % for thioalbumin-single mutant and 14 % for thioalbumin-double mutant (see Figure 48).

GE analysis revealed the ce of a small amount of high molecular weight species (HMWS) in all albumin-doxorubicin samples (see Figure 49). This data is consistent with that ed by SEC analysis (see Figure 50). In addition, GE analysis confirmed the conjugation of doxorubicin to albumin observed by UV-Vis spectroscopy (see Figure 49, DOX fluorescence) Albumin-doxorubicin and thioalbumin-doxorubicin conjugates were analysed along with non-conjugated albumin and non-conjugated thioalbumin controls by SEC chromatography. Analysis of the doxorubicin conjugates showed that the monomer content was approximately 97.6 % for albumin-DOX, 86.8 % for albumin-DOX (double ), (see Figure 50 B, D and F). Non-conjugated n and thioalbumin controls (see Figures 50 A, C and E) showed similar monomer and high molecular weight species content to the doxorubicin conjugated species. In particular, Figure 50 shows peaks at , (5003), (5005), (5007), (5009) and (5011) corresponding to the ce of the HMWS and peaks at (5002), (5004), (5006), (5008), (5010) and (5012) corresponding to the monomer content.

Both bumin mutants show a higher tendency to aggregate than wild-type albumin: 13.2-14.5 % for thioalbumin (single mutant) and 11.8-16.6 % for thioalbumin (double mutant), compared to 2.4-2.6 % for native albumin. Analysis of these samples confirmed that conjugation of doxorubicin via PL13-PEG4-val-citaminobenzoyl is well tolerated and produced no additional aggregation (see Figure 50 A, C and F).

Accordingly, it has been shown that the protein drug conjugates of the t invention provide suitable alternatives to known protein drug conjugates. Moreover, the linker incorporated into the protein drug conjugates of the present ion enables safe and effective drug delivery by successfully binding the drug to the protein and retaining the drug until the target tissue is reached.

Throughout this specification, unless the t requires otherwise, the word "comprise" or variations such as "comprises" or ising", will be understood to imply the inclusion of a stated integer or group of rs but not the exclusion of any other integer or group of integers.

Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in New Zealand or any other country.

Claims (15)

1. A protein drug conjugate formed by the reaction of a globular n, a linker and a drug, wherein the globular protein is an antibody or fragment thereof comprising at least one reactive thiol group and wherein the linker comprises a nitrogen containing heterocyclic aromatic ring comprising a vinyl substituent represented by the following formulae, 1 N R 2 (III) wherein: R1 is selected from; ? (CH2)n - C(O)- R, or ? (CH2)m – S – (CH2)n – C(O) – R, or ? (CH2)n - CH(COR)2, or, ? (CH2)m – S – (CH2)2CH(COR)2, or, wherein, - R2 is selected from the same group of molecules as R1, en, or an alkyl group, - n is any integer from 0 to 10, - m is any integer between 0 and 10, - R is a hydroxide (OH), amine or a poly-(alkylene ) group; wherein the globular protein is conjugated to the linker through the conjugation of the thiol group to the vinyl substituent; and wherein the drug is conjugated to the linker through the R1 group, the R2 group, or the R1 group and the R2 group.

2. The protein drug ate ing to claim 1, wherein the antibody is a monoclonal antibody.

3. The protein drug conjugate according to any preceding claim, wherein the drug is a cytotoxin or a therapeutic e or polypeptide.

4. The protein drug conjugate according to claim 3, wherein the cytotoxin is a biologically active cytotoxic al.

5. The protein drug conjugate according to claim 3 or claim 4, wherein the cytotoxin is an anticancer drug.

6. The protein drug conjugate according to any preceding claim, wherein the linker comprises a poly-(alkylene glycol) group.

7. The protein drug conjugate ing to claim 6, wherein the poly- ene glycol) group is a polyethylene glycol (PEG).

8. The protein drug conjugate according to claim 6 or claim 7, wherein the poly-(alkylene glycol) structure is provided with one or more reactive functional groups including hydroxy, amine, carboxylic acid, alkyl halide, azide, succinimidyl or thiol groups.

9. The protein drug conjugate according to any preceding claim, which further comprises an extender linker.

10. The protein drug conjugate according to claim 9, wherein said er linker is enzyme cleavable.

11. A method of producing the protein drug conjugate of any preceding claim, which comprises contacting the protein with the linker which is bound to the drug.

12. A method of producing the protein drug conjugate of any one of claims 1 to 10, which ses contacting the protein with the linker, and subsequently contacting the linker with the drug.

13. The method according to claim 11 or 12, including an initial step of reacting a precursor linker comprising a nitrogen containing heterocyclic aromatic ring having a vinyl substituent with a poly-(alkylene glycol) molecule to produce the linker.

14. The method according to any one of claims 11 to 13, further comprising an initial step of ing the protein to produce a variant polypeptide having a thiol group at one or more d positions of the polypeptide.

15. The drug n conjugate according to any one of claims 1 to 10 for use in therapy. Reactivity of Pill, 12 and 13 with giutathione x.\wx“ O. H. r?5. F. \L 1 1 hr \ct. 4L 3. . 9. .3 ”I. O. \\\\.&u¢.\$\i\: «..mx“ H C‘. . . a. .3:.. “x\\m 0. .3 7/ O. 0' \ct.1 2 \\