WO2005012349A2

WO2005012349A2 - Chromatographic process for the purification of notch ligands

Info

Publication number: WO2005012349A2
Application number: PCT/GB2004/003327
Authority: WO
Inventors: Bhupinder Dosanjh
Original assignee: Lorantis Limited
Priority date: 2003-08-01
Filing date: 2004-07-30
Publication date: 2005-02-10
Also published as: WO2005012349A3

Abstract

Methods for purifying Notch ligand proteins and fragments, variants and derivatives thereof are described.

Description

Process

Field of the invention

The present invention relates to purification of Notch ligand proteins and polypeptides, and derivatives, fragments and variants thereof.

Background of the invention

A Notch ligand protein or polypeptide is an agent capable of interacting with a Notch receptor to cause a biological effect. Particular examples of naturaly occurring mammalian Notch ligands identified to date include the Delta family, for example Delta or Delta-like 1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus noiyegicus and Genbank Accession No. NM__;016941 - Homo sapiens, and US 6121045 (Millennium)), Delta-4 (Genbank Accession Nos. AB043894 and AF 253468 - Homo sapiens) and the Serrate family, for example Serrate- 1 and Seιrate-2 (WO97/01571, O96/27610 and WO92/19734), Jagged-1 (Genbank Accession No. U73936 - Homo sapiens) and Jagged-2 (Genbank Accession No. AF029778 - Homo sapiens). Ho ology between family members is extensive.

Notch ligand proteins and polypeptides may be used to treat a variety of diseases, and are also useful as research tools for studying Notch signalling.

Although purification of Notch ligand proteins and polypeptides which have been

"tagged" with sequences such as IgFc and N5His domains is relatively straightforward by use of affinity reagents which bind to the "tag", there remains a need for purification methods which do not rely on use of such "tags" and which are thus capable of purifying an untagged Notch ligand protein or polypeptide, or fragment, derivative or variant thereof, especially from a cell culture supernatant or lysate resulting from expression of the protein or polypeptide in culture. A description of the Notch signalling pathway and conditions affected by it may be found, for example, in our published PCT Applications as follows:

PCT/GB97/03058 (filed on 6 November 1997 and pubhshed as WO 98/20142; claiming priority from GB 9623236.8 filed on 7 November 1996, GB 9715674.9 filed on 24 July 1997 and GB 9719350.2 filed on 11 September 1997);

PCT/GB99/04233 (filed on 15 December 1999 and published as WO 00/36089; claiming priority from GB 9827604.1 filed on 15 December 1999);

PCT/GB00/04391 (filed on 17 November 2000 and published as WO 0135990; claiming priority from GB 9927328.6 filed on 18 November 1999); PCT/GBOl/03503 (filed on 3 August 2001 and pubhshed as WO 02/12890; claiming priority from GB 0019242.7 filed on 4 August 2000);

PCT/GB02/02438 (filed on 24 May 2002 and pubhshed as WO 02/096952; claiming priority from GB 0112818.0 filed on 25 May 2001);

PCT/GB02/03381 (filed on 25 July 2002 and pubhshed as WO 03/012111; claiming priority from GB 0118155.1 filed on 25 July 2001);

PCT/GB02/03397 (filed on 25 July 2002 and pubhshed as WO 03/012441; claiming priority from GB0118153.6 filed on 25 July 2001, GB0207930.9 filed on 5 April 2002,

GB 0212282.8 filed on 28 May 2002 and GB 0212283.6 filed on 28 May 2002);

PCT/GB02/03426 (filed on 25 July 2002 and published as WO 03/011317; claiming priority from GBOl 18153.6 filed on 25 July 2001, GB0207930.9 filed on 5 April 2002,

GB 0212282.8 filed on 28 May 2002 and GB 0212283.6 filed on 28 May 2002);

PCT/GB02/04390 (filed on 27 September 2002 and published as WO 03/029293; claiming priority from GB 0123379.0 filed on 28 September 2001);

PCT/GB02/05137 (filed on 13 November 2002 and published as WO 03/041735; claiming priority from GB 0127267.3 filed on 14 November 2001 , PCT/GB02/03426 filed on 25 July 2002, GB 0220849.4 filed on 7 September 2002, GB 0220913.8 filed on

10 September 2002 and PCT/GB02/004390 filed on 27 September 2002);

PCT/GB02/05133 (filed on 13 November 2002 and published as WO 03/042246; claiming priority from GB 0127271.5 filed on 14 November 2001 and GB 0220913.8 filed on 10 September 2002). Each of PCT/GB97/03058 (WO 98/20142), PCT/GB99/04233 (WO 00/36089), PCT/GB00/04391 (WO 0135990), PCT/GBOl/03503 (WO 02/12890), PCT/GB02/02438 (WO 02/096952), PCT/GB02/03381 (WO 03/012111), PCT/GB02/03397 (WO 03/012441), PCT/GB02/03426 (WO 03/011317), PCT/GB02/04390 (WO 03/029293), PCT/GB02/05137 (WO 03/041735) and PCT/GB02/05133 (WO 03/042246) are hereby incorporated herein by reference.

Statements of Invention

According to the present invention there is provided a method for purifying a Notch ligand protein or polypeptide, or a fragment derivative or variant thereof, from a mixture comprising the steps of : i) contacting said mixture with a hydrophobic interaction chromatographic (HIC) support; and ii) selectively eluting the protein from the support.

Suitably the HIC support may be a C2-C20 alkyl- or an aryl- derivatised support; for example selected from C2-C12 alkyl agarose (the term "agarose" herein includes cross- linked agarose), aryl-agarose, C2-C12 alkyl sihca and aryl sihca; for example butyl-, phenyl-, or octyl- agarose.

Suitably the protein or polypeptide may be selectively eluted with a low salt buffer.

Suitably the eluting buffer may be a phosphate buffer, such as an alkali metal phosphate buffer, for example a sodium phosphate buffer.

For example, in one embodiment of the invention, the protein or polypeptide may be selectively eluted with a 10-200 mM sodium phosphate buffer, pH 6-9. Suitably the elution step comprises a step-wise increase in ionic strength, for example use of a linear gradient.

Suitably the method also comprises a step of salt precipitation, by use of salt such as ammonium sulphate.

Alternatively or in addition the method may further comprise a step (before or after the HIC step) of contact with an ion exchange support, followed by selective elution of the protein or polypeptide from the support.

Alternatively or in addition the method may further comprise a step (before or after the HIC step) of contact with a size exclusion support, followed by selective elution of the protein or polypeptide from the support.

Preferably the purification protocol should provide a protein or polypeptide product that is essentially free of other proteins, by which is meant at least 70%, preferably at least 80%, preferably at least 90% and preferably greater than 95% pure with respect to total protein in the preparation. The process will preferably also remove other contaminants or or reduce their presence to acceptable levels.

According to one aspect of the present invention there is provided a method for purifying a Notch ligand protein or polypeptide, or a fragment, derivative or variant thereof, from a mixture comprising the steps of (in either order): i) contacting the mixture with an ion exchange support (eg an anion exchange support) and selectively eluting the Notch ligand protein or polypeptide from the ion exchange support; and ii) contacting the mixture with a hydrophobic interaction chromatographic (HIC) support and selectively eluting the Notch ligand protein or polypeptide from the HIC support; According to a preferred aspect of the present invention there is provided a method for purifying a Notch ligand protein or polypeptide, or a fragment derivative or variant thereof, from a mixture comprising the steps of (in any order): i) filtering the mixture; ii) contacting the mixture with an ion exchange support (eg an anion exchange support) and selectively eluting the Notch ligand protein or polypeptide from the ion exchange support; iii) contacting the mixture with a hydrophobic interaction chromatographic (HIC) support and selectively eluting the Notch ligand protein or polypeptide from the HIC support; iv) contacting the mixture with a size exclusion chromatography support and selectively eluting the Notch ligand protein or polypeptide from the size exclusion chromatography support; and v) inactivating any viruses present.

According to another preferred aspect of the present invention there is provided a method for purifying a Notch ligand protein or polypeptide, or a fragment derivative or variant thereof, from a mixture comprising the steps of (in any order): i) filtering the mixture; ii) concentrating the mixture; iii) contacting the mixture with an ion exchange support (eg an anion exchange support) and selectively eluting the Notch ligand protein or polypeptide from the ion exchange support; iv) contacting the mixture with a hydrophobic interaction chromatographic (HIC) support and selectively eluting the Notch ligand protein or polypeptide from the HIC support; and v) contacting the mixture with a size exclusion chromatography support and selectively eluting the Notch ligand protein or polypeptide from the size exclusion chromatography support; and vi) inactivating any viruses. Suitably the Notch ligand protein or polypeptide may be Delta or Jagged, preferably Delta, eg Deltal , preferably human Deltal , or a fragment derivative or variant thereof.

Suitably the Notch ligand protein or polypeptide comprises a Notch ligand DSL domain and at least one Notch ligand EGF domain.

Suitably the Notch ligand protein or polypeptide comprises: i) a Notch ligand DSL domain; h) 1 -16 Notch ligand EGF domains ; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences.

Suitably the Notch ligand protein or polypeptide comprises: i) a Notch ligand DSL domain; ii) 1-8 Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences.

Suitably the Notch ligand protein or polypeptide comprises: i) a Notch ligand DSL domain; ii) 1-5 (and suitably not more than 5) Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences.

Suitably the Notch ligand protein or polypeptide comprises: i) a Notch ligand DSL domain; ii) 2-3 (and suitably not more than 3) Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-teπninal domain; and iv) optionally one or more heterologous amino acid sequences. Suitably the Notch ligand protein or polypeptide has at least 50% (suitably at least 70%, eg at least 80%, eg at least 90%) amino acid sequence similarity or identity to the following sequence along the entire length of the latter (SEQ ID NO:l):

MGSRCALA-^VLSALLCQV SS-^FELKLQEFVlWKGLLGlXπWCCRGGAGPPPCACRTF

FRVCLiiaiYQASVSPEPPeTYGSAVTPVLGVDSFSLPDGGG

TFSLIIEALHTDSPDDLATE PERLISRLATQRH TVGEE SQDLHSSGRTDLKYSYRF

VCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGF

CDKPGECKCRVG QGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHH KPCKNGATCTN GQGSYTCSCRPGYTGATCELGIDEG

According to a further aspect of the invention there is provided a preparation of an untagged Notch ligand protein or polypeptide (or fragment, variant or derivative thereof), obtainable by a method as described above, having at least 90% Notch hgand protein or polypeptide (or fragment, variant or derivative thereof) by weight ("untagged" here means essentially free of any heterologous "tag" sequence).

According to a further aspect of the invention there is provided a preparation of an untagged Notch ligand protein or polypeptide (or fragment, variant or derivative thereof) obtainable by a method as described above, having at least 95% Notch Hgand protein or polypeptide (or said fragment, variant or derivative thereof) by weight.

According to a further aspect of the invention there is provided a preparation of an untagged Notch ligand protein or polypeptide (or fragment, variant or derivative thereof) obtainable by a method as described above, having at least 90% Notch hgand protein or polypeptide (or said fragment, variant or derivative thereof) by weight, and less than 1 EU/mg protein of endo toxin; and less than 1 pg/mg protein DNA. In one embodiment the contact with the HIC support may be at about ambient ("room") temperature, ie typically around 23 °C.

In one embodiment the contact with the HIC support may be at a temperature of less than about 40°C, suitably from about 0 °C to about 40°C, for example from about 0 to about 20 °C, for example about 3 °C to about 10 °C, for example about 5 °C.

Typical conditions include a pH of from about 6 to about 8.5 and an ionic strength of from about 0.05 to 4.0M (eg expressed as NaCI).

Detailed description

Various preferred features and embodiments of the present invention will now be described in more detail by way of non-limiting example and with reference to the accompanying Figures, in which:

Figure 1 shows a schematic representation of the Notch signalling pathway;

Figure 2 shows schematic representations of the Notch ligands Jagged and Delta;

Figure 3 shows aligned amino acid sequences of DSL domains from various Drosophila and mammalian Notch ligands ;

Figure 4 shows the amino acid sequences of human Delta-1, Delta-3 and Delta-4;

Figure 5 shows the amino acid sequences of human Jagged-1 and Jagged-2;

Figures 6 to 8 show results from Example 2;

Figure 9 shows results of Example 3; and Figures 10-12 show further sample traces and gels.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabihties of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, M Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober (1992 and periodic supplements; Current Protocols in Immunology, John Wiley & Sons, New York, NY). Each of these general texts is hereby incorporated herein by reference.

For the avoidance of doubt, Drosophila and vertebrate names are used interchangeably and all homologues are included within the scope of the invention.

Hydrophobic Interaction Chromatography

As noted and discussed in US 5252216 (Folena-Wasserman , et al) hydrophobic molecules in a aqueous solvent will self-associate. This association is due to hydrophobic interactions. A hydrophobic hgand coupled to a matrix is variously referred to herein as an HIC support, HIC gel or HIC column. The strength of interaction between a protein and a HIC support is not only a function of the proportion of non-polar to polar surfaces on the protein but also the distribution of the non-polar surfaces.

A variety of matrices may be employed in the preparation of HIC columns, the most commonly used is agarose. However, other matrices such as silica and organic polymer resins may also be used. Useful hydrophobic ligands include but are not limited to alkyl groups having from about 2 to about 20, preferably from about 2 to 10 carbon atoms, for example from about 2 to 8 carbon atoms, such as a butyl, propyl, or octyl; or aryl groups such as phenyl. HIC products for gels and columns may be obtained commercially from suppliers such as Pharmacia LKB AB, Uppsala, Sweden under the product names butyl- Sepharose™, phenyl-Sepharose™, CL-4B , octyl-Sepharose FF ™ and phenyl-Sepharose EF ™; Tosoh Corporation, Tokyo, Japan under the product names Toyopearl Butyl 650M (Fractogel TSK Butyl-650) or TSK-GEL phenyl-5PW; Miles-Yeda, Rehovot, Israel under the product name alkyl-agarose, wherein the alkyl group contains from 2-10 carbon atoms, and J. T. Baker, PhiUipsburg, N.J. under the product name Bakerbond WP-HI- propyl.

As noted in US 5252216 the choice of a particular HIC support can be determined by the skilled worker. In general the strength of the interaction of the protein and the HIC hgand increases with the chain length of the of the alkyl ligands but ligands having from about 4 to about 8, eg from about 4 to 6 carbon atoms may be suitable. A phenyl group has about the same hydrophobicity as a pentyl group, although the selectivity can be quite different owing to the possibility of pi-pi interaction with aromatic groups on the protein.

Adsorption of the proteins to a HIC column is favored by high salt concentrations, but the actual concentrations can vary over a wide range depending on the nature of the protein and the particular HIC hgand chosen. Various ions can be arranged in a so-called soluphobic series depending on whether they promote hydrophobic interactions (salting- out effects) or disrupt the structure of water (chaotropic effect) and lead to the weakening of the hydrophobic interaction. Cations are typically ranked in terms of increasing salting out effect as B a⁺⁺ <Ca⁺⁺ <Mg^"^ <Li⁺ <Cs⁺ <Na⁺ <K⁺ <Rb⁺ <NH₄ ⁺. Anions may typically be ranked in terms of increasing chaotropic effect as PO₄ ^"" <SO₄ ^" <CH₃ COO^" <CT <Bf <NO₃< <ClO₄ ^" <I^" <SCN^". Accordingly, salts may be formulated that influence the strength of the interaction as given by the following relationship:

Na₂SO₄ >NaCl>(NH₄)₂SO₄ >NH₄Cl>NaBι NaSCN For example, salt concentrations of between about 0.75 and about 2M ammonium sulfate or between about 1 and 4M NaCI are useful (see eg US 5252216).

The influence of temperature on HIC separations is not simple, although generaUy a decrease in temperature decreases the interaction. However, any benefit that would accmeby increasing the temperature must also be weighed against adverse effects such an increase may have on the activity of the protein.

Elution, whether step wise or in the form of a gradient, can be accomplished in a variety of ways: (a) by changing the salt concentration, (b) by changing the polarity of the solvent or (c) by adding detergents. By decreasing salt concentration adsorbed proteins are eluted in order of increasing hydrophobicity. Changes in polarity may be affected by additions of solvents such as ethylene glycol or (iso)propanol thereby decreasing the strength of the hydrophobic interactions. Detergents function as displacers of proteins and have been used primarily in connection with the purification of membrane proteins.

HIC may be used in combination with other protein purification techniques. Thus it is preferred to apply HIC to material that has been partially purified by other protein purification procedures. By the term "partially purified" is meant a protein preparation in which the protein of interest is preferably present in at least 5 percent by weight, more preferably at least 10% and most preferably at least 45%.

Suitably the Notch hgand protein or polypeptide will be present in a mixture from a cell culture medium which has supported cell growth and/or cell maintenance and contains secreted product. A concentrated sample of such medium is preferably subjected to one or more protein purification steps prior to the application of a HIC step. For example, the sample may be subjected to ion exchange chromatography as a first step. As mentioned above various anionic or cationic substituents may be attached to matrices in order to form anionic or cationic supports for chromatography. Anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic exchange substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulosic ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K. Sephadex™ -based and cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP- Sephadex™ and DEAE-, Q-, CM- and S-Sepharose™ are all available from Pharmacia AB.

Because elution from ionic supports usually involves addition of salt and because, HIC is generally enhanced under increased s alt concentrations , the introduction of a HIC step following an ion exchange chromatographic step or other salt mediated purification step is particularly preferred. For example, an ion exchange chromatographic step and/or a salt (eg ammonium sulfate) precipitation step may suitably precede the application of HIC. Additional purification protocols may be added including but not necessarily limited to ion exchange chromatography, size exclusion chromatography, viral inactivation, concentration and freeze drying (US 5252216).

When the eluate resulting from HIC is subjected to ion exchange chromatography, both anionic and/or cationic procedures may be employed.

Additional Purification Steps

i) Gel Filtration Size Exclusion Chromatography

Size exclusion chiOmatography, otheiwise known as gel filtration or gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase into the pores of stationary phase particles. Differential penetration is a function of the hydrodynamic volume of the particles. Accordingly, under ideal conditions the larger molecules are excluded from the interior of the particles while the smaller molecules are accessible to this volume and the order of elusion can be predicted by the size of the protein because typically a linear relationship exists between elusion volume and the log of the molecular weight. Size exclusion chromatographic supports based on cross-linked dextrans, for example Sephadex ™, spherical agarose beads, for example Sepharose ™ (both commercially available from Pharmacia AB. Uppsala, Sweden), based on cross- linked polyacrylamides e.g. BIO-GEL ™ (commercially available from BioRad Laboratories, Richmond, Cahfomia) or based on ethylene glycol-methacrylate copolymer e.g. Toyopearl HW65S (commercially available from ToyoSoda Co., Tokyo, Japan) may suitably be used.

Suitably, a size exclusion chromatography step may be applied before or after the HIC step , preferably after the HIC step .

ii) Precipitation

Precipitation methods depend on the principle that in crude mixtures of proteins the solubilities of individual proteins are likely to vary widely. Although the solubihty of a protein in an aqueous medium depends on a variety of factors, a protein will generally be soluble if its interaction with the solvent is stronger than its interaction with protein molecules of the same or similar kind. Suitably, such a precipitation step may be applied before or after the HIC step, but preferably before the HIC step.

iii) Ion Exchange Chromatography

Ion exchange chromatography involves the interaction of charged functional groups in the sample with ionic functional groups of opposite charge on an adsorbent/matrix surface. Two general types of interaction are known. Anionic exchange chromatography mediated by negatively charged amino acid side chains (e.g. aspartic acid and glutamic acid side chains) interacting with positively charged surfaces and cationic exchange chromatography mediated by positively charged amino acid residues (e.g. lysine and arginine side chains and DEAE groups) interacting with negatively charged surfaces. Suitably, such an ion exchange step (eg anion exchange) may be applied before and/or after the HIC step, preferably before the HIC step.

The purified Notch hgand proteins and polypeptides obtained by the process of the invention preferably have the following properties:

1) greater than 95% Notch ligand protein or polypeptide by weight; 2) stable to proteolytic degradation at 4°C for at least three months; 3) low (<1 EU/mg protein) endotoxin; 4) low (<1 pg/mg protein) DNA; 5) non-Notch hgand protein or polypeptide <5% by weight; and 6) virally inactive.

Thus, according to a further aspect of the present invention there is provided a preparation of an untagged Notch ligand protein or polypeptide (or fragment, variant or derivative thereof) having at least 80%, preferably at least 90%, preferably at lest 95% Notch ligand protein or polypeptide by weight, and preferably less than 1 EU/mg proteinm of endotoxin; and preferably less than 1 pg DNA per mg protein.

Suitably, a Notch hgand protein or polypeptide suitable for purification in a method of the present invention may have at least 50%, preferably at least 70%, preferably at least 80%, preferably at least 90%, for example at least 95% amino acid sequence identity to the extracellular domain of the human Deltal sequence in Figure 4, over at least 250 amino acids of the latter, for example over substantially the entire length of the latter.

Suitably, a Notch hgand protein or polypeptide suitable for purification in a method of the present invention may have at least 50%, preferably at least 70%, preferably at least 80%, preferably at least 90%, for example at least 95% amino acid sequence identity to the extracellular domain of the human Delta3 sequence in Figure 4 over at least 250 amino acids of the latter, for example over substantially the entire length of the latter. Suitably, a Notch hgand protein or polypeptide suitable for purification in a method of the present invention may have at least 50%, preferably at least 70%, pieferably at least 80%, preferably at least 90%, for example at least 95% amino acid sequence identity to the extraceUular domain of the human Delta4 sequence in Figure 4 over at least 250 amino acids of the latter, for example over substantiaUy the entire length of the latter.

Suitably, a Notch hgand protein or polypeptide suitable for purification in a method of the present invention may have at least 50%, preferably at least 70%, pieferably at least 80%, preferably at least 90%, for example at least 95% amino acid sequence identity to the extraceUular domain of the human Jagged 1 sequence in Figure 5 over at least 250 amino acids of the latter, for example over substantiaUy the entire length of the latter.

Suitably, a Notch hgand protein or polypeptide suitable for purification in a method of the present invention may have at least 50%, preferably at least 70%, preferably at least 80%, preferably at least 90%, for example at least 95% amino acid sequence identity to the extraceUular domain of the human Jagged 2 sequence in Figure 5 over at least 250 amino acids of the latter, for example over substantiaUy the entire length of the latter.

Notch Ligand Proteins and Polypeptides

The term "Notch hgand" as used herein means an agent capable of interacting with a Notch receptor to cause a biological effect. The term as used herein therefore includes naturally occurring prcitein ligands such as Delta and Serrate/Jagged as weU as their fragments, homologues, variants and derivatives.

Particular examples of mammalian Notch ligands identified to date include the Delta family, for example Delta or Delta-like 1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus noiyegicus) and Delta-like 3 (Mus musculus) (Genbank Accession No. NM_016941 - Homo sapiens) and US

6121045 (MUlennium), Delta-4 (Genbank Accession Nos. AB043894 and AF 253468 - Homo sapiens) and the Serrate family, for example Serrate-! and Serrate-2 (WO97/01571, WO96/27610 and WO92/19734), Jagged-1 (Genbank Accession No. U73936 - Homo sapiens) and Jagged-2 (Genbank Accession No. AF029778 - Homo sapiens), and LAG-2. Homology between family members is extensive.

By a "homologue" is meant a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any one of the known Notch ligands, for example as mentioned above. Typically, a homologue of a known Notch ligand wiU be at least 20%, preferably at least 30%, identical at the amino acid level to the corresponding known Notch ligand (eg as shown in Figures 4 and 5 hereto) over a sequnce of at least 10, preferably at least 20, preferably at least 50, suitably at least 100 or at least 300 a ino acids, for example over substantiaUy the entire length of the extraceUular domain or over the entire length of the Notch ligand. Techniques and software for calculating sequence homology between two or more amino acid or nucleic acid sequences are well known in the art (see for example http://www.ncbi.nhn.nih.gov and Ausubel et al, Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.)

Notch ligands identified to date have a diagnostic DSL domain (D. Delta, S. Serrate, L. Lag2) comprising 20 to 22 amino acids at the amino terminus of the protein and up to 16 or more EGF-like repeats on the extraceUular surface. It is therefore preferred that homologues of Notch ligands also comprise a DSL domain at the N-terminus and up to 16 or more EGF- like repeats on the extraceUular surface.

Suitable homologues wUl be capable of binding to a Notch receptor. Binding may be assessed by a variety of techniques known in the art including in vitro binding assays.

Homologues of Notch ligands can be identified in a number of ways, for example by probing genomic or cDNA libraries with probes comprising all or part of a nucleic acid encoding a Notch hgand under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C). Alternatively, homologues may also be obtained using degenerate PCR which wiU generally use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences. The primers will contain one or more degenerate positions and wUl be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Polypeptide substances ma be purified from mammalian ceUs, obtained by recombinant expression in suitable host cells or obtained commerciaUy. Alternatively, nucleic acid constmcts encoding the polypeptides may be used. As a further example, overexpression of Notch or Notch ligand, such as Delta or Serrate, may be brought about by introduction of a nucleic acid construct capable of activating the endogenous gene, such as the Serrate or Delta gene. In particular, gene activation can be achieved by the use of homologous recombination to insert a heterologous promoter in place of the natural promoter, such as the Serrate or Delta promoter, in the genome of the target ceU.

Notch ligand domains

As discussed above, Notch ligands typically comprise a number of distinctive domains. Some predicted/potential domain locations for various naturally occurring human Notch ligands (based on amino acid numbering in the precursor proteins) are shown below:

Human Delta 1

Component Amino acids Proposed function/do:

SIGNAL 1-17 SIGNAL

CHAIN 18-723 DELTA- LIKE PROTEIN 1

DOMAIN 18-545 EXTRACELLULAR

TRANSMEM 546- 568 TRANSMEMBRANE

DOMAIN 569-723 CYTOPLASMIC

DOMAIN 159-221 DSL

DOMAIN 226-254 EGF-LIKE 1

DOMAIN 257 -285 EGF-LIKE 2

DOMAIN 292-325 EGF-LIKE 3

DOMAIN 332- 363 EGF-LIKE 4

DOMAIN 370-402 EGF-LIKE 5

DOMAIN 409-440 EGF-LIKE 6 DOMAIN 447-478 EGF-LIKE 7

DOMAIN 485-516 EGF-LIKE 8

HumanDelta 3

Component Amino acids Proposed function/doma

DOMAIN 158-248 DSL

DOMAIN 278-309 EGF-LIKE 1

DOMAIN 316-350 EGF-LIKE 2

DOMAIN 357-388 EGF-LIKE 3

DOMAIN 395-426 EGF-LIKE 4

DOMAIN 433-464 EGF-LIKE 5

HumanDelta 4

Component Amino acids Proposed function/do:

SIGNAL 1-26 SIGNAL

CHAIN 27-685 DELTA- IKE PROTEIN 4

DOMAIN 27-529 EXTRACELLULAR

TRANSMEM 530-550 TRANSMEMBRANE

DOMAIN 551-685 CYTOPLASMIC

DOMAIN 155-217 DSL

DOMAIN 218-251 EGF-LIKE 1

DOMAIN 252-282 EGF-LIKE 2

DOMAIN 284-322 EGF-LIKE 3

DOMAIN 324-360 EGF-LIKE 4

DOMAIN 362-400 EGF-LIKE 5

DOMAIN 402-438 EGF-LIKE 6

DOMAIN 440-476 EGF-LIKE 7

DOMAIN 480-518 EGF-LIKE 8

HumanJagged 1

Component Amino acids Proposed function/domaii

SIGNAL 1-33 SIGNAL

CHAIN 34-1218 JAGGED 1

DOMAIN 34-1067 EXTRACELLULAR

TRANSMEM 1068-1093 TRANSMEMBRANE

DOMAIN 1094-1218 CYTOPLASMIC

DOMAIN 167-229 DSL

DOMAIN 234-262 EGF-LIKE 1

DOMAIN 265-293 EGF-LIKE 2

DOMAIN 300-333 EGF-LIKE 3

DOMAIN 340-371 EGF-LIKE 4

DOMAIN 378-409 EGF-LIKE 5

DOMAIN 416-447 EGF-LIKE 6

DOMAIN 454-484 EGF-LIKE 7

DOMAIN 491-522 EGF-LIKE 8

DOMAIN 529-560 EGF-LIKE 9

DOMAIN 595-626 EGF-LIKE 10

DOMAIN 633-664 EGF-LIKE 11 DOMAIN 671-702 EGF-LIKE 12

DOMAIN 709-740 EGF-LIKE 13

DOMAIN 748-779 EGF-LIKE 14

DOMAIN 786-817 EGF-LIKE 15

DOMAIN 824-855 EGF-LIKE 16

DOMAIN 863-917 VON WILLEBRAND FACTOR C

HumanJagged 2

Component Amino acids Proposed funσtion/domai;

SIGNAL 1-26 SIGNAL

CHAIN 27-1238 JAGGED 2

DOMAIN 27-1080 EXTRACELLULAR

TRANSMEM 1081-1105 TRANSMEMBRANE

DOMAIN 1106-1238 CYTOPLASMIC

DOMAIN 178-240 DSL

DOMAIN 249-273 EGF-LIKE 1

DOMAIN 276-304 EGF-LIKE 2

DOMAIN 311-344 EGF-LIKE 3

DOMAIN 351-382 EGF-LIKE 4

DOMAIN 389-420 EGF-LIKE 5

DOMAIN 427-458 EGF-LIKE 6

DOMAIN 465-495 EGF-LIKE 7

DOMAIN 502-533 EGF-LIKE 8

DOMAIN 540-571 EGF-LIKE 9

DOMAIN 602-633 EGF-LIKE 10

DOMAIN 640-671 EGF-LIKE 11

DOMAIN 678-709 EGF-LIKE 12

DOMAIN 716-747 EGF-LIKE 13

DOMAIN 755-786 EGF-LIKE 14

DOMAIN 793-824 EGF-LIKE 15

DOMAIN 831-862 EGF-LIKE 16

DOMAIN 872-949 VON WILLEBRAND FACTOR C

DSL domain

A typical DSL domain may include most or all of the following consensus amino acid sequence (SEQ ID NO:2):

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys

Preferably the DSL domain may include most or all of the following consensus amino acid sequence:

Cys Xaa Xaa Xaa ARO ARO Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys BAS NOP BAS ACM ACM Xaa ARO NOP ARO Xaa Xaa Cys Xaa Xaa Xaa NOP Xaa Xaa Xaa Cys Xaa Xaa NOP ARO Xaa NOP Xaa Xaa Cys wherein:

ARO is an aromatic amino acid residue, such as tyrosfne, phenylalanine, tryptophan or histidine;

NOP is a non-polar amino acid residue such as glycine, alanine, proline, leucine, isoleucine or valine; BAS is a basic amino acid residue such as arginine or lysine; and

ACM is an acid or amide amino acid residue such as aspartic acid, glutamic acid, asparagine or glutamine. Preferably the DSL domain may include most or all of the following consensus amino acid sequence:

Cys Xaa Xaa Xaa Tyr yr Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Arg Pro Arg Asx Asp Xaa P e Gly His Xaa Xaa Cys Xaa Xaa Xaa Gly Xaa Xaa Xaa Cys Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Cys

(wherein Xaa may be any amino acid and Asx is either aspartic acid or asparagine).

An alignment of DSL domains from Notch ligands from various sources is shown in Figure 3.

The DSL domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human. Preferably the DSL domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch hgand sequence.

It wUl be appreciated that the term "DSL domain" as used herein includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturaUy occurring domains.

Suitably, for example, a DSL domain for use in a method of the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 1. Alternatively a DSL domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 2.

Alternatively a DSL domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 1.

Alternatively a DSL domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 3.

Alternatively a DSL domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 4.

EGF-like domain

The EGF-like motif has been found in a variety of proteins, as weU as EGF and Notch and Notch ligands, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518). For example, this motif has been found in extraceUular proteins such as the blood clotting factors IX and X (Rees et al., 1988, EMBO J. 7:2053- 2061; Furie and Furie, 1988, CeU 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766; Rothberg et al., 1988, CeU 55:1047-1059), and in some ceU- surface receptor proteins, such as thrombomodulin (Suzuki et al., 1987, EMBO J. 6:1891- 1897) and LDL receptor (Sudhof et al, 1985, Science 228:815-822). A protein binding site has been mapped to the EGF repeat domain in thrombomodulin and urokinase (Kurosawa et al., 1988, J. Biol. Che 263:5993-5996; AppeUa et al., 1987, J. Biol. Chem. 262:4437-4440).

As reported by PROSITE a typical EGF domain may include six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds. The main structure is proposed, but not necessarily required, to be a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines strongly vary in length as shown in the foUowing schematic representation of a typical EGF-like domain:

x(4) -C-x(0, 4S) -C-x(3, 12) -C-x(l , 70) -C-x(l, 6) -C-x(2) -G-a-x(0,ai) -α-5c(2 -C-ϊ I I ************************************

wherein:

'C: conserved cysteine involved in a disulfide bond. 'G': often conserved glycine 'a': often conserved aromatic amino acid '*': position of both patterns. 'x': any residue

The region between the 5th and 6th cysteine contains two conserved glycines of which at least one is normaUy present i ost EGF-like domains.

The EGF-like domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human. Preferably the EGF-like domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.

It wUl be appreciated that the term "EGF domain" as used herein includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturaUy occurring domains. Suitably, for example, an EGF-like domain for use in a method of the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 1.

Alternatively an EGF-like domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 2.

Alternatively an EGF-like domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 1.

Alternatively an EGF-like domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 3.

Alternatively an EGF-like domain for use in a method of the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, pieferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 4.

As a practical matter, whether any particular amino acid sequence is at least X% identical to another sequence can be determined conventionaUy using known computer programs. For example, the best overaU match between a query sequence and a subject sequence, also referred to as a global sequence alignment, canbe determined using a program such as the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are suitably both amino acid sequences. The result of the global sequence alignment is given as percent identity.

The term "Notch ligand N-terminal domain" means the part of a Notch ligand sequence from the N-teiminus to the start of the DSL domain. It wUl be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.

The term "heterologous amino acid sequence" or "heterologous nucleotide sequence" as used herein means a sequence (eg continuous sequence of at least 50 amino acids) which is not found in the native Notch hgand or its coding sequence.

Polypeptides, Proteins and Amino Acid Sequences

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "protein".

"Peptide" usually refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.

The amino acid sequence may be prepared and isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

Nucleotide Sequences

As used herein, the term "nucleotide sequence" is synonymous with the term ' 'polynucleotide" . The nucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin. They may also be cloned by standard techniques. The nucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.

Longer nucleotide sequences wiU generaUy be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This wiU involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. In general, primers wiU be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accompHshing this using automated techniques are readUy available in the art.

"Polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases in length and up to 5,000 bases or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

These may be constructed using standard recombinant DNA methodologies. The nucleic acid may be RNA or DNA and is preferably DNA. Where it is RNA, manipulations may be performed via cDNA intermediates. GeneraUy, a nucleic acid sequence encoding the first region will be prepared and suitable restriction sites provided at the 5' and/or 3' ends. Conveniently the sequence is manipulated in a standard laboratory vector, such as a plasmid vector based on pBR322 or pUC19 (see below). Reference may be made to Molecular Cloning by Sambrook et al. (Cold Spring Harbor, 1989) or similar standard reference books for exact detaUs of the appropriate techniques.

Sources of nucleic acid may be ascertained by reference to published literature or databanks such as GenBank. Nucleic acid encoding the desired first or second sequences may be obtained from academic or commercial sources where such sources are willing to provide the material or by synthesising or cloning the appropriate sequence where only the sequence data are avaUable. Generally this may be done by reference to literature sources which describe the cloning of the gene in question.

Variants, Derivatives, Analogues. Homologues and Fragments

In addition to the specific amino acid sequences mentioned herein, the present invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof.

In the context of the present invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. A variant sequence can be modified by addition, deletion, substitution modification replacement and/or variation of at least one residue present in the naturally-occurring protein.

The term "derivative" as used herein, in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide retains at least one of its endogenous functions. The term "analogue" as used herein, in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.

Within the definitions of "proteins" useful in the present invention, the specific amino acid residues may be modified in such a manner that the protein in question retains at least one of its endogenous functions, such modified proteins are referred to as "variants". A variant protein can be modified by addition, deletion and/or substitution of at least one amino acid present in the na raUy-occurring protein.

Typically, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required activity or abUity. Amino acid substitutions may include the use of non-naturally occurring analogues.

Proteins of use in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubihty, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues as long as the transport or modulation function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophflicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

For ease of reference, the one and three letter codes for the main naturaUy occurring amino acids (and their associated codons) are set out below: Symbol 3-letter Meaning Codons

A Ala Alanine GCT,GCC,GCA,GCG B Asp, Asn Aspartic, Asparagine GAT,GAC,AAT,AAC C Cys Cysteine TGT,TGC D Asp Aspartic GAT,GAC E Glu Glutamic GAA,GAG F Phe Phenylalanine τττ,ττc Θ Qly Glycine GGT,GGC,GGA,QGG H His Histidine CAT,CAC I He Isoleucine ATT,ATC,ATA K Lys Lysine AAA,AAG L Leu Leucine TTG,TTA,CTT,CTC,CTA,CTG M Met Methionine ATG N Asn Asparagine AAT,AAC P Pro Proline CCT,CCC,CCA,CCG Q Gin Qluta ine CAA,CAG R Arg Arginine CGT,CGC,CGA,CGG,AGA,AGG S Ser Serine TCT,TCC,TCA,TCG,AGT,AGC T Thr T reonine ACT,ACC,ACA,ACG V Val Valine GTT,GTC,GTA,GTG w Trp Tryptophan TGG X Xxx Unknown Y Tyr Tyrosine TAT, TAG z Glu, Gin Glutamic, Qlutamine GAA,GAG,CAA,GAG * End Terminator TAA,TAG,TGA

Conservative substimtions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

As used herein, the term "protein" includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms "polypeptide" and "peptide" refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. The terms subunit and domain may also refer to polypeptides and peptides having biological function.

"Fragments" are also variants and the term typicaUy refers to a selected region of the polypeptide or polynucleotide that is of interest either functionaUy or, for example, in an assay. "Fragment" thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

Such variants may be prepared using standard recombinant DΝA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DΝA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions wiU contain convenient restriction sites corresponding to sites in the nat- rally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DΝA ligated into the cut. The DΝA is then expressed in accordance with the invention to make the encoded protein. These methods are only nlustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

Polynucleotide variants will preferably comprise codon optimised sequences. Codon optimisation is known in the art as a method of enhancing RNA stabUity and therefor gene expression. The redundancy of the genetic code means that several different codons may encode the same amino-acid. For example, Leucine, Argfnine and Serrne are each encoded by six different codons. Different organisms show preferences in their use of the different codons. Viruses such as HJN, for instance, use a large number of rare codons. By changing a nucleotide sequence such that rare codons are replaced by the corresponding commonly used mammalian codons, increased expression of the sequences in mammalian target cells can be achieved. Codon usage tables are known in the art for mammalian ceUs, as weU as for a variety of other organisms. Preferably, at least part of the sequence is codon optimised. Even more preferably, the sequence is codon optimised in its entirety.

As used herein, the term "homology" can be equated with "identity". An homologous sequence wiU be taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical. In particular, homology should typically be considered with respect to those regions of the sequence known to be essential for an activity. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usuaUy, with the aid of readUy available sequence comparison programs. These commerciaUy available computer programs can calculate % homology between two or more sequences.

Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it faUs to take into consideration that, for example, in an otheiwise identical pair of sequences, one insertion or deletion wUl cause the foUowing amino acid residues to be put out of aUgnment, thus potentiaUy resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the aUgnment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - wiU achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaUer penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties wiU of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefor firstly requires the production of an optimal aUgnment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux).

Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410 (Atschul)) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are avaUable for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

The five BLAST programs available at http://www.ncbi.nlm.nih.gov perform the foUo wing tasks:

blastp - compares an amino acid query sequence against a protein sequence database.

blastn - compares a nucleotide query sequence against anucleoti.de sequence database.

blastx - compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.

tblastn - compares a protein query sequence against a nucleotide sequence database dynamically translated in aU six reading frames (both strands).

tblastx - compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

BLAST uses the following search parameters:

HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual). DESCRIPTIONS - Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).

EXPECT - The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match wiU not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).

CUTOFF - Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). TypicaUy, significance thresholds can be more intuitively managed using EXPECT.

ALIGNMENTS - Restricts database sequences to the number specified for which high- scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).

MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.

STRAND - Restrict a TBLASTN search to just the top or bottom strand of the database sequences ; or restrict a BLASTN, BL ASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence.

FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17 : 149 - 163 , or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). FUtering can eliminate statisticaUy significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted using the letter "N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") and the letter "X" in protein sequences (e.g., "XXXXXXXXX").

Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.

It is not unusual for nothing at aU to be masked by SEG, XNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect. NCBI-gi - Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.

Most preferably, sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.

In some aspects of the present invention, no gap penalties are used when determining sequence identity.

Although the final % homology canbe measured in terms of identity, the alignment process itself is typically not based on an aU-or-nothing pair comparison. Instead, a scaled similarity score matrix is generaUy used that assigns scores to each pairwise comparison based on chemical simUarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the pub he default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typicaUy does this as part of the sequence comparison and generates a numerical result.

Nucleotide sequences which are homologous to or variants of sequences of use in the present invention can be obtained in a number of ways, for example by probing DNA libraries made from a range of sources. In addition, other viral bacterial, or ceUular homologues particularly ceUular homologues found in mammalian ceUs (e.g. rat, mouse, bovine and primate ceUs), maybe obtained and such homologues and fragments thereof in general wiU be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA hbraries made from or genomic DNA hbraries from other animal species, and probing such hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and aUelic variants of the amino acid and/or nucleotide sequences useful in the present invention.

Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of use in the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence ahgnments canbe performed using computer software known in the art. For example the GCG Wisconsin PueUp program is widely used. The primers used in degenerate PCR wiU contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

As indicated above, with respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the reference sequences, preferably over the whole length of the reference sequences. More preferably there is at least 95%, more preferably at least 98%, homology. A suitable sequence comparison program is the GCG Wisconsin Bestfit program described above.

Cloning and Expression

Nucleotide sequences which are not 100% homologous to the sequences of the present invention but faU within the scope of the invention canbe obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA hbraries made from a range of sources, hi addition, other viral/bacterial, or ceUular homologues particularly ceUular homologues found in mammaUan ceUs (e.g. rat, mouse, bovine and primate ceUs), maybe obtained and such homologues and fragments thereof in general wiUbe capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA hbraries made from or genomic DNA hbraries from other animal species, and probing such hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency. SimUar considerations apply to obtaining species homologues and aUelic variants of the amino acid and/or nucleotide sequences useful in the present invention.

Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants homologues. Sequence ahgnments canbe performed using computer software known in the art. For example the GCG Wisconsin PUeUp program is widely used. The primers used in degenerate PCR wUl contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example sUent codon changes are required to sequences to optimise codon preferences for a particular host ceU in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the target protein or protein for T cell signalling modulation encoded by the nucleotide sequences.

The nucleotide sequences such as a DNA polynucleotides useful in the invention may be produced recombinantly, syntheticaUy, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers wiU be produced by synthetic means, involving a step wise manufacmre of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomphshing this using automated techniques are readfly avaUable in the art.

Longer nucleotide sequences wUl generaUy be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This wiU involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the amphfied fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amphfied DNA can be cloned into a suitable cloning vector

The present invention also relates to vectors which comprise a polynucleotide useful in the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides useful in the present invention by such techniques.

For recombinant production, host cells can be geneticaUy engineered to incorporate expression systems or polynucleotides of the invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals, such as Davis et al and Sambrook et al, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, baUistic introduction and infection. It wiU be appreciated that such methods can be employed in vitro or in vivo as drug delivery systems.

Representative examples of appropriate hosts include bacterial ceUs, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells; fungal ceUs, such as yeast ceUs and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 ceUs; animal cells such as CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce a polypeptide useful in the present invention. Such vectors include, among others, chromosomal, episomal and vims-derived vectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenovϊruses, fowl pox viruses, pseudorabies viruses and retro viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence maybe inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

Proteins or polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein or precursor. For example, it is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification. Likewise such an additional sequence may sometimes be desirable to provide added stability during recombinant production. In such cases the additional sequence may be cleaved (eg chemicaUy or enzymatically) to yield the final product. In some cases, however, the additional sequence may also confer a desirable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be prefeπed that the additional sequence is not removed so that it is present in the final product as administered.

Proteins or polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein or precursor. For example, it is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification. Likewise such an additional sequence may sometimes be desirable to provide added stabihty during recombinant production. In such cases the additional sequence may be cleaved (eg chemicaUy or enzymatically) to yield the final product. In some cases, however, the additional sequence may also confer a desirable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be prefeπed that the additional sequence is not removed so that it is present in the final product as administered. Arimi -itrafinn

Suitably the active agents ate administered in combination with a pharmaceuticaUy acceptable carrier or diluent. The pharmaceuticaUy acceptable carrier or dUuent may be, for example, sterile isotonic saline solutions, or other isotonic solutions such as phosphate- buffered saline. The conjugates of the present invention maybe admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubihsing agent(s). It is also prefeπed to formulate the compound in an oraUy active form.

Pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and wUl typically comprise any one or more of a pharmaceuticaUy acceptable diluent, carrier, or excipient. Acceptable carriers or dUuents for therapeutic use are weU known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or dUuent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubihsing agent(s).

Preservatives, stabUizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzo ate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

For some applications, active agents maybe administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents. Alternatively or in addition, active agents maybe administered by inhalation, intranasaUy or in the form of aerosol, or in the form of a suppository or pessary, or they may be applied topicaUy in the form of a lotion, solution, cream, ointment or dusting powder. An alternative means of transdermal administration is by use of a skin patch. For example, they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. They can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabhisers and preservatives as may be required.

TypicaUy, the physician wUl determine the actaal dosage which will be most suitable for an individual patient and it wiU vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such ate within the scope of this invention.

In general, a therapeuticaUy effective oral or intravenous dose is likely to range from 0.01 to 50 mg/kg body weight of the subject to be treated, preferably 0.1 to 20 mg/kg. The conjugate may also be administered by intravenous infusion, at a dose which is likely to range from 0.001-10 mg kg/hr.

Tablets or capsules may be administered singly or two or more at a time, as appropriate. It is also possible to administer active agents in sustained release formulations.

Active agents may also be injected parenteraUy, for example intracavernosally, intravenously, intramuscularly or subcutaneously

For parenteral administration, active agents may be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, agents may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

For oral, parenteral, buccal and sublingual administration to subjects (such as patients), the dosage level of active agents and their pharmaceuticaUy acceptable salts and solvates may typicaUy be from 10 to 500 mg (in single or divided doses). Thus, and by way of example, tablets or capsules may contain from 5 to 100 mg of active agent for administration singly, or two or more at a time, as appropriate.

The routes of administration and dosages described are intended only as a guide since a skUled practitioner wUl be able to detei ine readily the optimum route of administration and dosage for any particular patient depending on, for example, the age, weight and condition of the patient.

The term treatment or therapy as used herein should be taken to encompass diagnostic and prophylatic applications.

Various prefeπed features and embodiments of the present invention will now be described in more detail by way of non-limiting examples.

Example 1

i) Preparation of modulator of Notch signaUing in form of Notch hgand ExtraceUular domain fragment with free Cysteine tail for polymer coupling

A protein fragment comprising amino acids 1 to 332 (ie comprising DSL domain plus first 3 EGF repeats) of human Delta 1 (DLL-1 ; for sequence see GenBank Accession No AF003522) and ending with a free cysteine residue ("DlE3Cys") was prepared as foUows: A template containing the entire coding sequence for the extracellular (EC) domain of human DLL-1 (with two silent mutations) was prepared by a PCR cloning strategy from a placental cDNA library made from placental pofyA RNA (Clontech; cat no 6518-1) and combined with a C-terminal V5HES tag in a pCDNA3J plasmid (Invitrogen, UK) The template was cut HindUl to Pmel to provide a fragment coding for the EC domain and this was used as a template for PCR using primers as follows:

5'-primer: CAC CAT GGGCAGTCGGTGCGC GCT GG (SEQID NO:3)

3'-primer: GTC TAG GTT TAAACT TAACAC TCGTCAATC CCC AGC TCG CAG GTG (SEQ ID NO:4)

PCR was caπied out using Pfu turbo polymerase (Stratagene, La JoUa, CA, US) with cycling conditions as follows: 95C 5min, 95C lmin, 45-69C lmin, 72C lmin for 25 cycles, 72C lOrnin.

The products at 58C, 62C & 67C were purified from 1% agarose gel in 1 x TAE using a Qiagen gel extraction kit according to the manufacturer's instructions, ligated into pCRHblunt vector (InVitrogen TOPO-blunt kit) and then transformed into TOP10 ceUs (InVitrogen). The resulting clone sequence was verified, and only the original two silent mutations were found to be present in the parental clone.

The resulting sequence coding for "DlE3Cys" was excised using Pmel and Hind-H, purified on 1 % agarose gel, lx TAE using a Qiagen gel extraction kit and ligated into pCDNA3.1 V5HIS (Invitrogen) between the Pmel and HindDI sites, thereby eliminating the V5HIS sequence. The resulting DNA was transformed into TOP10 cells. The resulting clone sequence was verified at the 3'-ligation site.

The DlE3Cys-coding fragment was excised from the pCDNA3J plasmid using Pmel and Hindm. A pEE14.4 vector plasmid (Lonza Biologies, UK) was then restricted using EcoRI, and the 5 '-overhangs were filled musing Klenow fragment polymerase. The vector DNA was cleaned on a Qiagen PCR purification column, restricted using Hindm, then treated with Shrimp Alkaline Phosphatase (Roche). The pEE14.4 vector and DlE3cys fragments were purified on 1% agarose gel in 1 x TAE using a Qiagen gel extraction kit prior to ligation (T4 ligase) to give plasmid pEE14.4 DLLΔ4-8cys. The resulting clone sequence was verified.

The DlE3Cys coding sequence is as foUows (SEQ ID NO: 5):

1 atgggcagtc ggtgcgcgct ggccctggcg gtgctctcgg ccttgctgtg

51 tcaggtctgg agctctgggg tgttcgaact gaagctgcag gagttcgtca

101 acaagaaggg gctgctgggg aaccgcaact gctgccgcgg gggcgcgggg

151 ccaccgccgt gcgcctgccg gaccttcttc cgcgtgtgcc tcaagcacta

201 ccaggccagc gtgtcccccg agccgccctg cacctacggc agcgccgtca

251 cccccgtgct gggcgtcgac tccttcagtc tgcccgacgg cgggggcgcc

301 gactccgcgt tcagcaaccc catccgcttc cccttcggct tcacctggcc

351 gggcaccttc tctctgatta ttgaagctct ccacacagat tctcctgatg

401 acctcgcaac agaaaaccca gaaagactca tcagccgcct ggccacccag

451 aggcacctga cggtgggcga ggagtggtcc caggacctgc acagcagcgg

501 ccgcacggac ctcaagtact cctaccgctt cgtgtgtgac gaacactact

551 acggagaggg ctgctccgtt ttctgccgtc cccgggacga tgccttcggc

601 cacttcacct gtggggagcg tggggagaaa gtgtgcaacc ctggctggaa

651 agggccctac tgcacagagc cgatctgcct gcctggatgt gatgagcagc

701 atggattttg tgacaaacca ggggaatgca agtgcagagt gggctggcag

751 ggccggtact gtgacgagtg tatccgctat ccaggctgtc tccatggcac

801 ctgccagcag ccctggcagt gcaactgcca ggaaggctgg gggggccttt

851 tctgcaacca ggacctgaac tactgcacac accataagcc ctgcaagaat

901 ggagccacct gcaccaacac gggccagggg agctacactt gctcttgccg

951 gcctgggtac acaggtgcca cctgcgagct ggggattgac gagtgttaa

The DNA was prepared for stable cell line transfection/selection in a Lonza GS system using a Qiagen endofree maxi-prep kit. ii) Expression of DlE3Cys

Linearisation of DNA

The pEE14.4 DLLΔ4-8cys plasmid DNA from (i) above was linearised by restriction enzyme digestion with Pvul, and then cleaned up using phenol chloroform isoamyl alcohol (IAA), followed by ethanol precipitation. Plasmid DNA was checked on an agarose gel for linearisation, and spectrophotometry was used at 260/280nm for quantity and quality of preparation.

Transfection

CHO-K1 cells were seeded into 6 weUs at 7.5 x 10⁵ cells per weU in 3ml media (DMEM 10% FCS) 24hrs prior to transfection, giving 95% confluency on the day of transfection. Lipofectamine 2000 was used to transfect the ceUs using 5ug of linearised DNA. The transfection mix was left on the cell sheet for 5 Vι hours before replacing with 3ml semi- selective media (DMEM, 10% dFCS, GS) for overnight incubation. At 24 hours post-transfection the media was changed to fuU selective media (DMEM (Dulbecco's Modified Eagle Medium), 10%dFCS (fetal calf serum), GS (glutamine synthase), 25uM L-MSX (methionine sulphoximine)) and incubated further.

Cells were plated into 96 weUs at 10 ceUs per weU on days 4 and 15 after transfection.

96 well plates were screened under a microscope for growth 2 weeks post clonal plating. Single colonies were identified and scored for % confluency. When colony size was >30% media was removed and screened for expression by dot blot against anti-human- Delta-1 antisera. High positives were confirmed by the presence of a 36kDa band reactive to anti-human-Delta-1 antisera in PAGE Western blot of media.

Cells were expanded by passaging from 96 weU to 6 well to T25 flask before freezing. The fastest growing positive clone (LC09 0001) was expanded for protein expression. DlE3Cys expression and purification

T500 flasks were seeded with lx 10⁷ ceUs in 80ml of selective media. After 4 days incubation the media was removed, cell sheet rinsed with DPBS and 150ml of 325 media with GS supplement added to each flask. Flasks were incubated for 7 further days before harvesting. Harvest media was filtered through a 0.65- 0.45um filter to clarify prior to freezing. The amino acid sequence of the resulting expressed DlE3Cys Notch ligand protein was as follows:

MGSRCALALAVLS-^ C VWSSGVFELK QEFVNKKGLLGKKNCCRGGAGPPPCACRTF

FRVCLKHYQASVSPEPPCTYGSAVTPV GVDSFS PDGGGADSAFSNPIRFPFGFTWPG

TFS IIEALHTDSPDD ATENPER ISRLATQRHLTVGEEWSQD HSSGRTDLKYSYRF VCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPIC PGCDEQHGF

CD-^GECKCRVGWQGRYCDECI YPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHH

KPC GATCTNTGQGSYTCSCRPGYTGATCE GIDEC

(wherein the sequence in italics is the leader peptide, the underlined sequence is the DSL domain, the bold sequences are the three EGF repeats, and the terminal Cys residue is shown bold underlined).

Example 2

Purification of expressed DlE3Cvs b HIC

AU the following steps were carried out at 4°C. Notch ligand protein harvests from Example 1 above were clarified by 0.45μm filtration, pooled into 10 litre batches and then concentrated 10-fold by tangential flow filtration (TFF). The concentrate was then diluted with an equal volume of 2M ammonium sulphate and adjusted to pH8 and a final concentration of 1M ammonium sulphate. The product was then subjected to Hydrophobic Interaction Chromatography (HIC), using a 24 ml Butyl Sepharose 4FF (Amersham Pharmacia) column with gradient elution against 50mM sodium phosphate, pH8.0.

Butyl Sepharose 4FF is an aliphatic (butyl) hydrophobic interaction chromatography medium constructed from highly cross-linked 90 μm agarose beads derivatized via uncharged, chemicaUy stable ether linkages. A trace of the progress of elution is shown in Figure 6. The eluate was concentrated and buffer exchanged using centrifugal concentrators according to the manufacturers' instmctions. The purity of the product was determined by SDS PAGE. Sample gels are shown in Figure 7 and 8.

Example 3

The process of Example 2 was modified by use of a step elution rather than gradient elution. In a first procedure, elution was carried out using IM to 0M ammonium sulphate gradient spread over 20 column volumes. In an alternative procedure, elution was carried out using IM ammonium sulphate stepped directly to 0M. Elution traces are shown for comparison in Figure 9.

Example 4

The procedure of Example 2 was repeated with the modification that instead of using centrifugal concentrators the eluate was subjected to size exclusion chromatography in order to separate the monomer from higher molecular weight contaminants. The monomer-containing fractions were pooled and concentrated and filter sterilised (0-2 μm).

Further sample traces and gels are shown in Figures 10 to 12.

Claims

CLAEV1S

1. A method for purifying a Notch ligand protein or polypeptide, or a fragment, derivative or variant thereof, from a mixture comprising the steps of : i) contacting said mixture with a hydrophobic interaction chromatographic (HIC) support; and ii) selectively eluting the protein, polypeptide, or fragment, derivative or variant thereof, from the support.

2. A method as claimed in claim 1 wherein the HIC support is a C2-C20 alkyl or an aryl derivatised support.

3. A method as claimed in claim 2 wherein the HJC support is a C2-C12 alkyl derivatised support.

4. A method as claimed in claim 3 wherein the HIC support is a C2-C8 alkyl derivatised support.

5. A method as claimed in claim 1 wherein the HIC support is selected from C2-C12 alkyl agarose; aryl-agarose; C2-C12 alkyl silica; and aryl sUica.

6. A method as claimed in claim 2 wherein the support is selected from butyl-, phenyl-, and octyl- agarose.

7. A method as claimed in any one of the preceding claims wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted with a low salt buffer.

8. A method as claimed in claim 7 wherein the eluting salt is an ammonium salt.

9. A method as claimed in claim 7 or claim 8 wherein the eluting buffer is an alkaU metal phosphate.

10. A method as claimed in any one of the preceding claims wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted at a pH of from about pH 4 to about pH 10.

11. A method as claimed in claim 10 wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted at a pH of from about pH 6 to about pH 10.

12. A method as claimed in claim 11 wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted at a pH of from about pH 7 to about pH 9.

13. A method as claimed in claim 12 wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted about pH 8.

14. A method as claimed in any one of the preceding claims wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted with a 10-200 mM phosphate buffer, pH 6-8 containing 0.01 to 5 M of an ammonium salt.

15. A method as claimed in claim 14 wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted with a 10-200 mM alkali metal phosphate buffer, pH 6-8 containing 0.01 to 3 M of an ammonium salt.

16. A method as claimed in claim 15 wherein the protein or polypeptide (or fragment, derivative or variant thereof) is selectively eluted with a 10-200 mM sodium phosphate buffer, pH 6-8 containing 0.01 to 2 M ammonium sulfate.

17. A method as claimed any of the preceding claims wherein the elution comprises a step-wise increase in ionic strength.

18. A method as claimed in claim 17 wherein the elution step comprises the use of a linear gradient.

19. A method as claimed in any of the preceding claims further comprising a step of salt precipitation.

20. A method as claimed in claim 19 wherein the salt is ammonium sulphate.

21. A method as claimed in any of the preceding claims further comprising a step of ion exchange chromatography, before or after the HIC step.

22. Amethod as claimed in any of the preceding claims further comprising a step of size exclusion chromatography, before or after the HIC step.

23. A method as claimed in any of the preceding claims wherein the Notch ligand protein or polypeptide is human Delta or Jagged or a fragment, derivative or variant thereof.

24. A method as claimed in any of the preceding claims wherein the Notch ligand protein or polypeptide is human Delta 1 , 3 or 4 or Jagged 1 or 2 or a fragment, derivative or variant thereof.

25. A method as claimed in any of the preceding claims wherein the Notch ligand protein or polypeptide is human Delta 1 or a fragment, derivative or variant thereof.

26. A method as claimed in any of the preceding claims wherein the Notch ligand protein or polypeptide (or fragment, derivative or variant thereof) comprises a Notch hgand DSL domain and at least one Notch ligand EGF domain.

27. A method as claimed in any of the preceding claims wherein the Notch hgand protein or polypeptide (or fragment, derivative or variant thereof) comprises: i) a Notch ligand DSL domain; ii) 1-5 (and suitably not more than 5) Notch hgand EGF domains; iii) optionaUy all or part of a Notch ligand N-teπninal domain; and iv) optionaUy one or more heterologous amino acid sequences.

28. A method as claimed in any of the preceding claims wherein the Notch ligand protein or polypeptide (or fragment, derivative or variant thereof) comprises: i) a Notch ligand DSL domain; ii) 2-4 (and suitably not more than 4) Notch hgand EGF domains; hi) optionaUy all or part of a Notch hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences.

29. A method as claimed in any of the preceding claims wherein the Notch ligand protein or polypeptide (or fragment, derivative or variant thereof) comprises: i) a Notch ligand DSL domain; ii) 2-3 (and suitably not more than 3) Notch hgand EGF domains; hi) optionaUy all or part of a Notch hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences.

30. A method as claimed in any of the preceding claims wherein the Notch ligand protein or polypeptide (or fragment, derivative or variant thereof) has at least 50% amino acid sequence similarity to the foUowing sequence along the entire length of the latter (SEQ JD NOJ):

MGSRCALAILAV1,SAL CQVWSS(^FKLK Q FRVCLIOIYQASVSPEPPCTYGSAVTPV G ΓØSFSLPDGGGADSAFSNPIIIPPFGFT PG TFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRF VCDEHYYGEGCSVFCRPRDDAFGHFTCGERGE VCNPGWKGPYCTEPICLPGCDEQHGF CDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGG FCNQD NYCTHH KPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDEC

31. A prep aration of an untagged Notch hgand protein or polypeptide (or fragment, variant or derivative thereof) obtainable by a method as claimed in any of claims 1 to 29 having at least 90% Notch hgand protein or polypeptide (or fragment, variant or derivative thereof) by weight.

32. A preparation of an untagged Notch hgand protein or polypeptide (or fragment, variant or derivative thereof) as claimed in claim 31 having at least 95% Notch hgand protein or polypeptide (or said fragment, variant or derivative thereof) by weight.

33. A preparation of an untagged Notch hgand protein or polypeptide (or fragment, variant or derivative thereof) as claimed in claim 31 having at least 90% Notch hgand protein or polypeptide (or said fragment, variant or derivative thereof) by weight, and less than 1 EU/mg protein of endotoxin; and less than 1 pg/mg protein DNA.