The compositions are useful in a variety of applications, in particular the medical field, in the agrochemical field and in the field of paper processing.

BACKGROUND AND PRIOR ART It is known that drugs can be targeted to specific sites.

Thus, efforts have been made to increase the target specificity of various drugs and modify their release characteristics e. g., rapid release, delayed release, sustained release or a combination thereof. In some cases, a particular cell type present in a diseased tissue or organ may express a unique cell surface marker. In such a case, an antibody can be raised against the unique cell surface marker and a drug can be linked to the antibody. Upon administration of the drug/antibody complex to the patient, the binding of the antibody to the cell surface marker results in the delivery of a relatively high concentration of the drug to the diseased tissue or organ.

Similar methods can be used where a particular cell type in the diseased organ expresses a unique cell surface receptor or a ligand for a particular receptor. In these cases, the drug can be linked to the specific ligand or to the receptor, respectively, thus providing a means to deliver a relatively high concentration of the drug to the diseased organ.

Cellulose binding domains ("CBDs") have been described as useful agents for the attachment of molecular species to cellulose, and have shown to be effective purification tools (US 5738984, US 6124117). WO 94/24158 discloses a nucleic acid sequence encoding a CBD as well as a fusion protein comprising the CBD and a second protein, and the potential use of such molecules, including drug delivery, affinity separations, and diagnostic techniques. However, such steps disclosed when coupled to drugs involve the chemical conjugation of the drug to the CBD. This represents an additional cost implication and may result in unfavorable economics in terms of ratios of CBD molecules to drug molecules (at least one CBD per drug molecule would be required). Furthermore, the chemical coupling process itself may negatively affect the drug, and the number of drug molecules that can be targeted is limited.

EP-A-0381719 discloses the formation of a polysaccharide matrix using fusion proteins having the substrate binding region of a cellulase which is lacking in cellulase activity.

Targeted delivery using cellulose binding domains has been previously described in an unrelated field of the art. WO 01/46357 discloses detergent compositions which are capable of delivering a benefit agent to a fabric during a washing or rinsing process. Such compositions comprise a fusion protein comprising a cellulose binding domain and a domain having a high binding affinity for another ligand. The fusion protein is bi-functional in its binding ability, whereby the cellulose binding domain region binds to cellulosic based materials and the second domain binds to another ligand.

WO 98/00500 discloses detergent compositions wherein a benefit agent is delivered onto fabric during the wash cycle by means of a peptide or protein deposition aid having high affinity for fabric, for example the cellulose binding domain of a cellulase enzyme, to which it is attached or absorbed. The benefit agent is a fabric softening agent, perfume, photoprotective agent, dye fixative agent, and the like.

Antibodies have also been shown to be effective in targeting liposomes to tumor cells (Bendas G. Biodrugs 15 (4): 215-224 (2001) ), but this relies on the incorporation of anti-bodies into the outer surface/membrane of the particles known also as immuno-liposomes.

There remains a need for systems that can deliver a benefit agent (e. g. , for pharmaceutical or agrochemical purposes) in a relatively high amount compared to the amount of antibody that is used.

There is also a need for systems that do not require covalent bonding of the antibody to the benefit agent.

Our copending international application, entitled"fusion proteins for delivery of therapeutic and other agents and compositions containing them", relates to the delivery of micro-capsules or micro-particles using proteins.

The present invention aims to provide'compositions for the delivery of micro-capsules or micro-particles containing a protein comprising a carbohydrate binding domain and a selective binding domain (eg, an antibody or fragment thereof) in which the protein has good stability and is relatively easy to produce.

SUMMARY OF THE INVENTION According to the present invention in a first aspect, there is provided a composition or kit of parts comprising: a micro- particle or micro-capsule ; and a protein which comprises a first binding domain which is a carbohydrate binding domain and a second binding domain which is capable of selective binding, wherein the first or second binding domain is capable of binding to said microcapsule or microparticle and the protein exhibits polysaccharidase activity.

Another aspect of the invention is a method of delivering a pharmaceutical or agrochemical benefit agent to a site in a patient or in or on a plant which comprises treating the patient or plant with a composition or kit of parts of the invention.

A further aspect of the invention is a pharmaceutical composition or kit of parts of the invention for use in medicine.

Yet another aspect of the invention is the use of a pharmaceutical composition or kit of parts of the invention in the manufacture of a medicament for the treatment of a condition in which the benefit agent is useful.

In another aspect, the invention provides a sterile'article for use in medicine which has been treated with a composition or kit of parts of the invention.

The invention also provides a method of forming the sterile article of the invention which comprises treating a substrate with a composition or kit of parts of the invention.

Another aspect of the invention is a method of treating paper which comprises applying to the paper a composition according to the invention.

Yet another aspect of the invention is a paper product which has been treated with a composition or kit of parts of the invention.

These and other aspects of the present invention will be explained hereinafter in some more detail.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the principle of the present invention showing as a preferred embodiment a self assembling targeted controlled release system using a polysaccharide binding domain-antibody fusion protein or conjugate.

DETAILED DESCRIPTION OF THE INVENTION The Protein The invention is based on the use of a protein having polysaccharidase activity. The protein can comprise a polysaccharidase conjugated or fused to the second binding domain (eg, an antibody or fragment thereof), in which case at least one of the carbohydrate binding domains in the protein is derived from the polysaccharidase. In another embodiment, the

carbohydrate binding domain is separate from the polysaccharidase activity, for example being provided from a different enzyme eg, the protein may comprise, in addition to. the second binding domain, a carbohydrate binding domain from one enzyme fused or conjugated to a polysaccharidase, or the active part thereof, from a different enzyme.

It is believed that using a polysaccharidase as part of the protein can improve the stability of the protein compared to a corresponding protein in which the protein comprises only the polysaccharide (ie, carbohydrate) binding domain of the polysaccharidase, without the polysaccharidase activity.

Moreover, the use of the polysaccharidase can result in the protein being easier to produce by fusion or conjugation methods known in the art and can provide more sites for the attachment of further binding domains, such as the second binding domain.

Preferably, the carbohydrate binding domain is capable of recognizing a useful binding site or a microcapsule or micro- particle containing at least one benefit agent. A preferred carbohydrate binding domain is a cellulose binding domain.

Preferably, the second binding domain is an antibody or fragment thereof. More preferably, the antibody or fragment thereof is a Heavy Chain antibody obtainable from the group of Camelidae. The protein may comprise one or more further optional binding domains. Preferably, the further optional binding domain (s) of the protein of the invention is a binding domain which is capable of binding to a therapeutic agent, a therapeutically useful binding site or a molecule or compound associated with it (for example, a molecule or substance put into the surface of a micro-capsule or a molecule complexed or otherwise associated with said therapeutically useful binding

site to enable the binding domain to recognise it) or, more preferably, a micro-particle or micro-capsule containing a high payload of at least one therapeutically useful agent. In some circumstances more than one binding domain will be able to recognise a disease site or otherwise therapeutically useful binding site.

Preferred polysaccharide binding domains include cellulose binding domains obtainable from a fungal enzyme origin such as Humicola, Trichoderma, Thermomonospora, Phanerochaete, Aspergillus or from a bacterial enzyme origin such as Bacillus, Clostridium, Streptomyces, Cellulomonas and Pseudomonas.

Suitable sources of cellulose binding domains are disclosed in US 6331416, the contents of which are incorporated by reference herein.

The protein may further comprise one or more additional binding domains i. e. , in addition to the first and second binding domains. For example, the protein may comprise a third binding domain which is capable of binding to said micro-particle or micro-capsule and/or a binding domain which is capable of binding to a therapeutic agent or a molecule or compound associated with it. Also, the protein may comprise two or more of the first binding domains and/or two or more of the second binding domains.

It will be understood that there is not intended to be any significance attached herein to the words first, second, third and related terms in connection with the definition of the binding domains, other than for the purposes of identifying different parts or regions of the protein. Thus, the first and second binding domains can be present in the protein in any order and the protein may be produced from any genetic format

(for example, the first binding domain need not necessarily be formed first nor be the major part of the molecule).

In one embodiment of the invention, a protein comprises an antibody (or other targeting moiety) which recognises a specific disease site (e. g. , cancer cells), and a carbohydrate<BR> binding domain (e. g. , cellulose binding domain) that recognises<BR> and binds to a particulate vehicle (e. g. , a micro-capsule) that comprises at least one therapeutic or pharmaceutically useful compound whose structure or coating contains the carbohydrate recognised by the carbohydrate binding domain. The therapeutic advantage of this construct is that disease site-specific delivery can be combined with, e. g. , a micro-capsule containing a relatively high payload of at least one therapeutic agent.

This is different from other attempts at high payload site specific delivery as they have been limited by either the number of therapeutic molecules that can be linked to a single antibody or require the targeting moiety (e. g. antibody) to be incorporated into the surface of the micro-capsule such as in the case of immuno-liposomes.

In another embodiment of the invention, the proteins facilitate the biphasic delivery of therapeutic agents from the same delivery vehicle. This aspect is represented diagrammatically in Figure 1. Biphasic delivery of therapeutic agents and pharmaceutical excipients has previously been shown to enhance the therapeutic effect of pharmaceutically useful compounds, such as propranolol in clinical trials (Barnwell SG et al, 1996, International Journal of Pharmaceutics, 128,145-154).

Biphasic delivery includes, for example, the release of the same therapeutic agent under two separate conditions, for example rapid and sustained release or alternatively the release of two or more therapeutic agents at different rates or

under different external conditions, for example both rapid release or both sustained release or one rapid release and one sustained release or pH dependent release. Biphasic release may be achieved by using a protein containing a carbohydrate binding domain bound to a micro-capsule comprising a carbohydrate recognised by the binding domain of the protein and at least one therapeutic agent together with a micro- capsule together with an additional binding domain recognising and binding to a micro-capsule containing at least one therapeutic agent (e. g. , an anti-body recognising the gelatine comprising a gelatine micro-capsule, or another moiety on a capsule surface). Release (e. g. , rapid release, sustained release, pH dependent release) of the therapeutic agent (s) from each of the two bound micro-capsules is usually determined by the composition of the micro-capsules.

In a further embodiment of the present invention, a protein is provided that combines the advantages of each of the delivery vehicles described above. In this instance, the protein is furnished with at least two additional binding domains in addition to the carbohydrate binding domain. This type of construct enables the disease site-specific delivery of a biphasic release delivery vehicle containing at least one therapeutic agent. Such fusion variants contain more than two protein domains specified such that the fusion contains at least one carbohydrate binding domain and at least one protein domain exhibiting a binding affinity to another molecule or surface.

In still another embodiment of this invention, the use of the carbohydrate-binding domain of the protein as a targeting moiety is envisaged. In this case, the carbohydrate binding domain is applied to bind a surface containing a carbohydrate,

for example a paper product, a cellulose-containing dressing, a medical device, or plant leaves, stems or roots, whereas at least one other domain on the protein is used to bind a micro- capsule containing a therapeutic agent useful in pharmaceutical or agrochemical applications.

The present invention is advantageous over the prior art in that it is capable of greatly increasing the amounts of a compound that can be delivered using a single protein by the use of micro-capsules or micro-particulate constructs that comprise a high payload of at least one benefit agent.

It will be appreciated that in the compositions and kit of parts of the invention, the first or second binding domain is capable of binding to said microcapsule or microparticle in order that the microparticle can be delivered. Thus, either the first binding domain is capable of binding to the microcapsule or microparticle and the second binding domain is targeted to the intended site of delivery (e. g. , an antibody directed to a particular cell surface antigen) or the first binding domain directs the delivery of the protein (e. g. , binding to a carbohydrate-containing substrate such as the surface of a plant) and the second binding domain is capable of binding to the microcapsule or microparticle.

The proteins and compositions according to the present invention are prepared by methods which are well known in the art. The proteins are preferably produced as fusion proteins and can be prepared, for example, starting from the disclosures of WO 01/46357 and/or WO 98/00500 and a skilled person will have no difficulty in making any modification or addition thereof based on the teaching of the present specification and

his knowledge of the art. The same applies to the preparation of the compositions of the present invention.

The proteins of the invention may also be prepared by conjugation of one part of the protein to one or more other parts of the protein. For example, the protein can be prepared by the formation of one or more covalent bonds between different peptides using a suitable linker molecule according to techniques that are well known in the art. As an example, the second binding domain (eg, an antibody or antibody fragment) can be chemically conjugated to a polysaccharidase, having a carbohydrate binding domain, to form the protein of the invention.

The Carbohydrate Binding Domain In one aspect, the present invention relates to a protein comprising a carbohydrate binding domain and at least one other domain having a high binding affinity for another ligand. The latter preferably comprises a micro-capsule or construct comprising a high payload (i. e. , preferably more than 10, more preferably more than 100, even more preferably more than 1,000, most preferably more than 10,000 molecules per micro-capsule, microparticle or construct) of at least one therapeutic agent or optionally a disease site or otherwise therapeutically useful binding site where the carbohydrate binding domain may be used either to bind a micro-particle containing a high payload of at least one therapeutically useful agent or optionally bind to the surface of a therapeutically useful surface such as a bandage, dressing or medical device.

Cellulose Binding Domains mentioned before are examples of polysaccharide binding domains (PBDs) which can also be referred to as Carbohydrate Binding Domains. The term carbohydrate binding domain also includes protein sequences such as Starch Binding Domains, Mannose Binding Domains, Xylan Binding Domains, and Chitin Binding Domains. Examples of the range of these polysaccharide binding domains available are described by Henrissat B. , Davies G. J. (Plant Physiology (2000) 124 (4): 1515-1519) and in US6331416, the contents of both documents being incorporated by reference herein. The term Carbohydrate Binding Domain will be collectively used hereinafter for any of such binding domains which can be used as part of the protein according to the present invention, unless indicated otherwise, and may also be referred to as CBD.

A preferred binding domain is the Cellulose Binding Domain which will be used hereinafter as a typical example of the carbohydrate binding domain part of the protein described and claimed in the present invention. Thus, the abbreviation CBD is used hereinafter to indicate Carbohydrate Binding Domains in general, whereas a Cellulose Binding Domain is a typical and preferred example of the broader definition of Carbohydrate Binding Domains so that no confusion will arise.

A Cellulose Binding Domain is a polypeptide which has high affinity for or binds to water soluble or water insoluble forms of cellulose and chitin, including crystalline forms. Cellulose Binding Domains are found as integral parts of large protein complexes consisting of two or more different polypeptide domains, for example in hydrolytic enzymes (hydrolases) which typically are composed of a catalytic domain containing the active site for substrate hydrolysis, and a Carbohydrate Binding Domain such as a Cellulose Binding Domain for binding

to the insoluble matrix. Such enzymes can comprise more than one catalytic domain and one, two or three CBDs and optionally one or more polypeptide regions linking the CBD (s) with the catalytic domain (s), the latter regions usually being denoted a "linker". Examples of hydrolytic enzymes comprising a CBD are cellulases, xylanases, mannanases, arabinofuranosidases, acetyl esterases and chitinases. CBDs have also been found in algae, e. g. the red alga Porphyra purpurea as a non-hydrolytic polysaccharide binding protein, see Peter Tomme et al., "Cellulose Binding Domains : Classification and Properties"in "Enzymatic Degradation of Insoluble Carbohydrates", John N.

Saddler and Michael H. Penner (Eds. ), ACS Symposium Series, No.

618,1996. However, most of the known CBDs are from cellulases and xylanases.

In this context, the term"Cellulose Binding Domain"is intended to be understood as defined by Tomme et al. , op. cit.

This definition classifies more than 120 Cellulose Binding Domains into 10 families (I-X) which may have different functions or roles in connection with the mechanism of substrate binding. However, it is anticipated that new family representatives and additional CBD families will appear in the future.

A carbohydrate binding domain may be exemplified by a binding domain recognising cellulose. A cellulose binding domain is a part of many cellulolytic enzymes and can be obtained therefrom. Cellulose binding domains are also obtainable from xylanase and other hemicellulase degrading enzymes. Preferably, the cellulose binding domain is obtainable from a fungal enzyme origin such as Humicola, Trichoderma, Thermomonospora, Phanerochaete, Aspergillus or from a bacterial enzyme origin such as Bacillus, Clostridium, Streptomyces, Cellulomonas and

Pseudomonas. Other polysacharide binding sites of interest include those that recognise the polysacharide components of glycoproteins found within animal tissues.

The second and possibly further binding domains In the protein according to the invention, the carbohydrate binding domain is fused to a second and, optionally, a third binding domain having high binding affinities for other ligands, respectively. Preferably, the carbohydrate binding domain (exemplified by a cellulose binding domain) is connected to the domain having a high binding affinity for another ligand by means of a linker consisting of about 0-20, preferably about 2-15, more preferably of 2-5 amino acid residues.

The second, optional third and any other binding domains may be derived from any molecules or fragments of molecules which are capable of exhibiting selective binding affinity, preferably they are peptides or proteins. The second domain having a high binding affinity for another ligand usually is an antibody or antibody fragment, although the invention is not limited to antibodies and antibody fragments, and other molecules having selective binding affinity may be used instead. Especially preferred are heavy chain antibodies such as found in Camelidae.

The protein according to the invention may comprise more than two recognition domains. It is for example possible to produce a CBD protein with more than one antibody domain, in which the antibody domains may bind to the same or bind to different antigens. Conversely, it is also possible in the CBD antibody fusion format to produce a molecule with one antibody domain with more than one CBD, whereby the CBDs incorporated may be

identical sequences or may be isolated from more than one source, or modified varieties thereof.

The protein is comprised of two or more binding domains whereby at least one binding domain is a carbohydrate binding domain and at least one domain having a high binding affinity for another ligand or molecule, such as an antibody domain or a fragment derived thereof.

Generally speaking, the degree of binding of a molecule A to another molecule B can be generally expressed by the chemical equilibrium constant Kd resulting from the following reaction: [X] + [B] > fX = By The chemical equilibrium constant Kd is then given by: Kd = (AJxLBJ<BR> fX=B] Whether the binding of a molecule to the target site/ligand is specific or not can be judged from the difference between the binding (Kd value) of the molecule to one type of target site/ligand, versus the binding to another type of non-target site ligand material. For pharmaceutical and agrochemical applications, said ligand material will form part of or be associated with a therapeutic agent, preferably in a micro- capsule or other construct. In this aspect of the invention, the CBD region of the protein binds to a carbohydrate containing material and the high affinity domain region binds to the therapeutic agent construct. Alternatively, this approach can be reversed whereby the therapeutic site is targeted by the high affinity domain of the protein and the carbohydrate binding domain region binds to a carbohydrate based or carbohydrate containing therapeutic agent construct or

micro-capsule. However, it will usually be more convenient to measure Kd values and differences in Kd values on other materials such as a polystyrene microtitre plate or a specialised surface in an analytical biosensor. The difference between the two binding constants should be minimally 10, preferably more than 100, and more preferably, more than 1000.

Typically, the reagent should bind to the target site/ligand, with a Kd lower than 10-3 M, preferably lower than 10-6M and could be 10-9 M or even less. Higher binding affinities (Kd of less than 10-5 M) and/or a larger difference between the one type of target-site/ligand and another type of target site/ligand (or background) binding would increase the deposition of the benefit agent. In summary, improved delivery of therapeutic agents in addition to potentially improving therapeutic efficacy would also offer the opportunity to lower the dose of the therapeutic agent while maintaining efficacy.

Several classes of agents or molecules can be envisaged which are capable of binding to a specific targeted site, for example in a diseased tissue or organ. As stated before, an important and preferred class of such agents or molecules is the class of antibodies which will be discussed below in more detail. Other classes, such as, e. g. peptides and peptidomimics, been mentioned and discussed in WO 01/46357 and WO 98/00500 the contents of which are herein incorporated by reference. The use of such agents or molecules is also contemplated in the present invention.

Antibodies

The second binding domain which is capable of selective binding is preferably an antibody or an antibody fragment. Antibodies are specific binding proteins.

Antibodies are well known examples of molecules which are capable of binding specifically to compounds against which they were raised. Antibodies can be derived from several sources.

From mice, monoclonal antibodies can be obtained which possess very high binding affinities. From such antibodies, Fab, Fv or scFv fragments, can be prepared which have retained their binding properties. Such antibodies or fragments can be produced through recombinant DNA technology by microbial fermentation. Well known production hosts for antibodies and their fragments are yeast, moulds or bacteria.

A class of antibodies of particular interest is formed by the Heavy Chain antibodies as found in Camelidae, such as the camel or the llama. The binding domains of these antibodies consist of a single polypeptide fragment, namely the variable region of the heavy chain polypeptide (HC-V). In contrast, in the classic antibodies (murine, human, etc. ), the binding domain consists of two polypeptide chains (the variable regions of the heavy chain (Vh) and the light chain (Vl)). Procedures to obtain heavy chain immunoglobulins from Camelidae, or (functionalized) fragments thereof, have been described in WO-A-94/04678, and WO-A-94/25591, and are incorporated herein by reference.

Alternatively, binding domains can be obtained from the Vh fragments of classical antibodies by a procedure termed "camelization". Hereby the classical Vh fragment is transformed, by substitution of a number of amino acids, into a HC-V-like fragment, whereby its binding properties are retained. This procedure has been described by Riechmann et al. in a number of publications (J. Mol. Biol. (1996) 259,957-969 ; Protein. Eng.

(1996) 9,531-537, Bio/Technology (1995) 13,475-479). Also HC- V fragments can be produced through recombinant DNA technology in a number of microbial hosts (bacterial, yeast, mould), as described in WO-A-94/29457.

Methods for producing proteins that comprise an enzyme and an antibody or that comprise an enzyme and an antibody fragment are already known in the art. One approach is described by Neuberger and Rabbits (EP-A-0 194 276). A method for producing a protein comprising an enzyme and an antibody fragment that was derived from an antibody originating in Camelidae is described in WO-A-94/25591. A method for producing bispecific antibody fragments is described by Holliger et al. (1993) PNAS 90, 6444-6448.

A particularly attractive feature of antibody binding behaviour is their reported ability to bind to a"family"of structurally related molecules. For example, in Gani et al. (J. Steroid Biochem. Molec. Biol. 1994 48,277-282) an antibody is described that was raised against progesterone but also binds to the structurally-related steroids, pregnanedione, pregnanolone and 6-hydroxy-progesterone.

Where antibodies are used to target therapeutic agents internally (for example intravenous, intra-muscular or even following oral administration) the proteins may be prepared in a humanised form to prevent the stimulation of an immune response in the recipient.

Antibodies suitable for use in the invention include antibodies used in cancer treatments. Examples include Rituximab (Rituxan/Mab Thera) and alemtuzumab (Mab-Compath), marketed by Roche/Genentech and Schering respectively, IDEC's ibritumab

tiuxetan (Zevalin), trastuzumab (Herceptin) a mouse monoclonal antibody treatment used to treat advanced breast cancers that over expresses HER2 protein, marketed by Roche, and gemtuzumab- ozogamicin (Mylotarg) for the treatment of acute myeloid leukemia. Other suitable antibodies include those used for the treatment of rheumatoid arthritis including Schering-Plough/ Centocor's Infliximab (Remicade) and Wyeth's entanercept (Enbrel) which recognise and block the action of TNF (Tumour Necrosis Factor) and Amgen's anakinra (Kineret) which works through the inhibition of interleukin 1 activity. Further examples are antibodies used in transplantation and control of immunity, such as monoclonal antibody treatments used to prevent T-lymphocyte proliferation are used in the prophylaxis of acute rejection in allogenic renal transplantation including basiliximab (Simulect) and daclizumab (Zonapax) marketed by Novartis and Roche, respectively.

The Polysaccharidase The proteins of the invention have polysaccharidase activity.

Of particular interest are those polysaccharidases that are capable of hydrolysing glucan chains. Examples of polysaccharidases are those molecules that degrade polysaccharides such as cellulose, xylan, starch or chitin eg, cellulases, xylanases, amylases or chitinases respectively.

Preferably, the protein has cellulase activity. By this, we mean that the protein has the capability of hydrolysing cellulose (beta-1, 4-glucan linkages) to form molecules of lower molecular weight than cellulose, such as glucose, cellobiose, cello-oligosaccharides, etc.

The term cellulase, as used herein, includes enzymes selected from exoglucanases or exocellobiohydrolases, endoglucanases, and beta-glucosidases. These three different types of cellulase enzymes act synergistically to convert cellulose and its derivatives to glucose. The cellulase may be of bacterial, plant or fungal origin.

The cellulase enzyme of the invention can be obtained from any cellulase producing microorganism. The cellulase may have a cellulose binding domain, in addition to and separate from the carbohydrate binding domain in the protein of the invention, or it may lack a cellulose binding domain. Cellulases which lack a cellulose binding domain are known in the art. Naturally- occurring cellulases which lack a cellulose binding domain include, for example, bacterial cellulases derived from Erwinia carotovora (see, e. g. , Saarilahti et al. Gene, Vol. 90, pp. 9-<BR> 14 (1990) ) and Clostridium thermocellum (see, e. g. , Gilkes et<BR> al., Microbiological Reviews, pp. 303-315 (1991) ). Cellulases are preferably bacterial, plant and fungal cellulases. More preferably, the cellulases are fungal cellulases, most preferably derived from Trichoderma sp. , including Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii, Penicillium sp. , Humicola sp. , including Humicola insolens, Aspergillus sp. and Fusarium sp. As used herein, the term "Trichoderma"or"Trichoderma sp. "refers to any fungal strains which have previously been classified as Trichoderma or which are currently classified as Trichoderma. Bacterial cellulases from Thermomonospora sp. , Cellulomonas sp., Bacillus sp. ,<BR> Pseudomonas sp. , Clostridium sp. and Streptomyces sp. are also preferred.

The polsaccharidase may be produced by recombinant techniques.

Genetically modified cellulases which are derived from a DNA sequence in which all or part of the nucleotides in the coding region which encodes the cellulose binding domain have been deleted, replaced or otherwise manipulated to destroy the cellulose binding characteristics are also considered within the scope of the invention. Such modified cellulases are described in PCT Publication No. WO 95/16782.

Compositions The systems of the present invention may be in the form of a composition e. g. , a homogeneous or non-homogeneous mixture of the protein and the micro-particles or microcapsules. The system may alternatively be in the form of a kit of parts which comprises the protein packaged separately from the micro- particles or microcapsules; the protein and the micro-particles or microcapsules may be mixed before use or used separately i. e. , the protein and the micro-particles or microcapsules may be intended for simultaneous, separate or sequential use. When the system of the invention is a kit of parts, it preferably comprises a two part package, typically together with instructions for use.

Pharmaceutical Compositions The compositions of the invention may be pharmaceutical compositions in which the micro-particle or micro-capsule comprises a pharmaceutical benefit agent.

By"pharmaceutical benefit agent", as used herein, we mean any suitable agent which can be used for benefitting-for human welfare-an organism or tissue or surface to be treated or

intended to be treated. Such benefit agents include, for example, therapeutic agents, in particular relatively small organic molecules, peptides, proteins, DNA, RNA, vaccines, vectors used in gene therapy, live or dead micro-organisms, or any other substance or system used to treat or prevent a disease state in plants or animals, including humans. Thus, when the composition or kit of parts is for pharmaceutical use, the benefit agent is preferably selected from therapeutic small molecules, peptides, proteins, nucleic acids, vaccines and vectors used in gene therapy.

Therapeutic agents which may be delivered include, for example, proteins, peptides, nucleic acids and small organic molecules, for example local anesthetics (such as cocaine, procaine and lidocaine), hypnotics and sedatives (such as barbiturates, benzodiazepines and chloral derivatives), psychiatric agents (such as phenothiazines, tricyclic antidepressants and monoamine oxidase inhibitors), anti-epilepsy compounds (such as hydantoins), L-dopa, opium-based alkaloids, analgesics, anti- inflammatories, allopurinol, cancer chemotherapeutic agents, anticholinesterases, sympathomimetics (such as epinephrine, salbutamol and ephedrine), antimuscarinics (such as atropine), ß-adrenergic blocking agents (such as phentolamine), 5- adrenergic blocking agents (such as propranolol), ganglionic stimulating and blocking agents (such as nicotine), neuromuscular blocking agents, autacoids (such as anti- histamines and 5-HT antagonists), prostaglandins, plasma kinins (such as bradykinin), cardiovascular drugs (such as digitalis), antiarrhythmic drugs, antihypertensives, vasodilators (such as amyl nitrate and nitroglycerin), diuretics, oxytocin, antibiotics, anthelminthics, fungicides, antiviral compounds (such as acyclovir), anti-trypanosomals, anticoagulants, sex hormones (for example for HRT or contraception), insulin,

alprostidil, blood-clotting factors, calcitonin, growth hormones, vaccines, constructs for gene therapy and steroids.

The recipient may be a human or any other vertebrate, preferably a mammal, bird or fish for example a cow, sheep, horse, pig, chicken, turkey, dog, cat or salmon, or a plant, especially for DNA transformation of the plant.

Conditions in which the benefit agent may be useful for treatment or prophylaxis or for the alleviation of symptoms include, for example, bacterial infections, viral infections, cancer, CNS disorders, cardiovascular disease and allergic diseases. Specific examples of conditions include diabetes, genetic disorders (treated by gene therapy), pain, addiction, insomnia and sleep disorders, eating disorders, epilepsy, psychiatric disorders, Parkinson's disease, Alzheimer's disease, depression, stroke, rheumatic diseases, asthma, eczema, HIV and AIDS, gastric disorders and hypertension.

Agricultural Compositions Another preferred form of compositions of the invention is an agricultural composition in which the micro-particle or micro- capsule comprises an agricultural benefit agent.

The term"agricultural benefit agent", as used herein, is defined in an analogous manner to the term"pharmaceutical benefit agent"and thus includes any suitable agent which can be used for directly or indirectly benefiting-for plant welfare-a plant or plant tissue or plant surface to be treated or intended to be treated.

When the composition or kit of parts of the invention is an agricultural composition or kit of parts, the benefit agent is preferably selected from pesticides (e. g. , insecticides), herbicides, fungicides, plant growth stimulating agents, insect attractants or repellents (such as pheromones) and crop protecting agents.

Examples of agrochemicals suitable for use in the invention include glyphosate, fomesafen, glufosinate, mecoprop p, methylchlorophenoxy acetic acid, propamocarb, fosetyl, triasulfuron, tribenuron, metasulfuron, thifensulfuron, flupysulfuron, iodosulfuron, rimsulfuron, nicosulfuron, cinosulfuron, bensulfuron, trifloxysulfuron, flumetsulam, metosulam, chloransulam, floransulam, imazethabenz, imazethapyr, imazaquin, imazamox, flucarbazone, propoxycarbazine, amicarbazone, clodinafop, fenoxaprop, diclofop, propaquizafop, quizalofop, fluazifop, cyhalofop, haloxyfop, sethoxydim, clethodim, tralkoxydim, dicamba, clopyralid, 2,4-D, fluroxypyr, amicarbazone, azafenidin, benfluamid, benzfendizone, benzobicyclon, cinidon-ethyl, diclosulam, fentrazamid, flufenacet, flufenpyr, foramsulphuron, indanofan, mesosulphuron, oxaziclomefone, penoxsulam, pethoxamid, picolinafen, profoxidim, profluazol, propoxycarbazone, pyraflufen, pyrazogyl, sulphosulphuron, tepraloxydim, tritosulphuron and analogues and/or agrochemically acceptable salts thereof.

Paper Treatment Compositions Another preferred composition of the invention is a paper treatment composition. In compositions that are suitable for the treatment of paper, the micro-capsule or micro-particle

comprises a material that is beneficial in the processing of paper or in the final paper product.

The compositions of the invention have the advantage that they can assist in the degradation of the paper product, when used, and/or assist in the processing of pulp formed from the paper, as a result of their polysaccharidase activity. This can be particularly advantageous if the polysaccharidase (eg, cellulase) has a greater activity in the presence of water than in the absence of water, so that the polysaccharidase becomes more effective in degrading the paper when the paper is wet.

In the manufacture of paper products, it is often desirable to enhance physical and/or optical properties by the addition of chemical additives. Typically, chemicals such as softeners, colorants, brighteners, strength agents, etc. are added to the fiber slurry upstream of the headbox in a paper making machine during the manufacturing or converting stages of production to impart certain attributes to the finished product.

The term"paper"is used herein to broadly include writing, printing, wrapping, sanitary, and industrial papers, newsprint, linerboard, tissue, bath tissue, facial tissue, napkins, wipers, wet wipes, towels, absorbent pads, intake webs in absorbent articles such as diapers, bed pads, meat and poultry pads, feminine care pads, and the like made in accordance with any conventional process for the production of such products.

With regard to the use of the term"paper"as used herein includes any web containing cellulosic fibers alone or in combination with other fibers, natural or synthetic. It can be layered or unlayered, creped or uncreped, and can consist of a single ply or multiple plies. In addition, the paper or tissue

web can contain reinforcing fibers for integrity and strength.

The material contained in the microcapsule or microparticle in the paper treatment compositions of the invention can be, for example, one or more than one additive selected from: dry strength aids, wet strength aids, softening agents, adsorbency aids, sizing agents, dyes, optical brighteners, chemical tracers, opacifiers, dryer adhesive chemicals, and the like.

Additional forms of additives may include: pigments, emollients, humectants, viricides, bactericides, buffers, waxes, fluoropolymers, odor control materials and deodorants, zeolites, perfumes, vegetable and mineral oils, humectants, sizing agents, surfactants, moisturizers, UV blockers, antibiotic agents, lotions, fungicides, preservatives, aloevera extract, vitamin E, or the like. Suitable additives are preferably adsorbable by the papermaking fibers.

The term"softening agent"refers to any adsorbable chemical additive that can be incorporated into paper products such as tissue to provide improved tactile feel and reduce paper stiffness. A softening agent may be selected from the group consisting of quaternary ammonium compounds, quaternized protein compounds, phospholipids, silicone quaternaries, quaternized, hydrolyzed wheat protein/dimethicone phosphocopolyol copolymer, organoreactive polysiloxanes, and silicone glycols. These adsorbable chemical additives can also act to reduce paper stiffness or can act solely to improve the surface characteristics of tissue, such as by reducing the coefficient of friction between the tissue surface and the hand.

The method for applying adsorbable chemical additives to the pulp fibers may be used in a wide variety of pulp finishing processing, including dry lap pulp, wet lap pulp, crumb pulp, and flash dried pulp operations. By way of illustration, various pulp finishing processes (also referred to as pulp processing) are disclosed in Pulp and Paper Manufacture: The Pulping of Wood, 2Ed., Volume 1, Chapter 12. Ronald G.

MacDonald, editor, which is incorporated by reference.

All known papermaking fibers may be used, including bleached and unbleached fibers, fibers of natural origin (including wood fiber and other cellulose fibers, cellulose derivatives, and chemically stiffened or crosslinked fibers), some component portion of synthetic fiber (synthetic papermaking fibers include certain forms of fibers made from polypropylene, acrylic, aramids, acetates, and the like), virgin and recovered or recycled fibers, hardwood and softwood, and fibers that have been mechanically pulped (e. g. , groundwood), chemically pulped (including but not limited to the kraft and sulfite pulp processings), thermomechanically pulped, chemithermomechanically pulped, and the like. Mixtures of any subset of the above mentioned or related fiber classes may be used. The pulp fibers can be prepared in a multiplicity of ways known to be advantageous in the art.

The protein of the present invention can impart an antimicrobial effect on account of its polysaccharidase activity.

Formulations and Carriers

In the compositions of the invention, the protein and the microparticles or the microcapsules are formulated together.

In the kit of parts, the protein and the microparticles or the microcapsules are formulated separately (i. e. , as separate formulations).

Compositions and formulations of the invention may comprise a suitable carrier. Preferred compositions and formulations are pharmaceutical compositions where the carrier is a pharmaceutically acceptable carrier either in solid or in liquid form. The carrier contains, for example, recognised pharmaceutical excipients, such as thickeners, solvents, pH regulators, emulsifiers or cryogenic stabilizing agents. Such carriers are widely known in the art and a skilled person will have no difficulty in selecting an appropriate carrier for making a pharmaceutical composition or formulation of the invention, taking into consideration the intended use and/or wishes of the prescriber, usually a skilled person in the medical field, or user. Likewise, similar compositions and formulations are contemplated for agrochemical use where the protein of the invention carries a high payload of a benefit agent of choice, such as a growth-stimulating or crop- protecting agent or a herbicide or pesticide, and the carrier is a suitable carrier for such use, e. g. on crops, either in solid or, preferably, in liquid form, e. g. water or another suitable solvent. Again, a skilled person will have no difficulty in selecting an appropriate carrier depending on the specific use of the protein according to the invention. If desired, the active ingredient of the compositions and formulations of the invention can be brought in a form which facilitates its processing or uptake by the living organism, e. g. a suitable salt or ester.

In therapeutic use, the compositions, the formulations or the protein or the microparticles or microcapsules may, for example, be administered orally, rectally, parenterally or topically. Thus, the therapeutic compositions and formulations of the present invention may take the form of any of the known pharmaceutical compositions for oral, rectal, parenteral or topical administration. Pharmaceutically acceptable carriers suitable for use in such compositions are well known in the art of pharmacy. The compositions and formulations of the invention may contain 0. 1-99% by weight of the protein and the microparticles or microcapsules. The compositions and formulations of the invention will generally be prepared in unit dosage form. The excipients used in the preparation of these compositions are known to the skilled person.

Compositions and formulations for oral administration include, for example tablets, capsules, syrups and aqueous or oil suspensions. The excipients used in the preparation of these compositions are known in the art. Tablets may be prepared using an inert diluent such as calcium phosphate in the presence of disintegrating agents, for example maize starch, and lubricating agents, for example magnesium stearate, and tableting the mixture by known methods. The tablets may be formulated in a manner known to those skilled in the art so as to give a sustained release of the protein and/or microcapsules of the present invention. Such tablets may, if desired, be provided with enteric coatings by known methods, for example by the use of cellulose acetate phthalate. Similarly, capsules, for example hard or soft gelatin capsules, containing the protein and/or the microcapsules with or without added excipients, may be prepared by conventional means and, if desired, provided with enteric coatings in a known manner. The

tablets and capsules may conveniently each contain 1 pg to 500 mg of the active compound. Other compositions for oral administration include, for example, aqueous suspensions containing the active compound in an aqueous medium in the presence of a non-toxic suspending agent such as sodium carboxymethylcellulose, and oily suspensions containing a compound of the present invention in a suitable vegetable oil, for example arachis oil.

Dosage forms suitable for oral administration may comprise tablets, pills, capsules, caplets, multiparticulates including: granules, beads, pellets and micro-encapsulated particles; powders, elixirs, syrups, suspensions and solutions.

Pharmaceutical compositions and formulations may also be administered parenterally (for example subcutaneously, intramuscularly, intradermally and/or intravenously [such as by injection and/or infusion] ) in the known pharmaceutical dosage forms for parenteral administration (for example sterile suspensions in aqueous and/or oily media and/or sterile solutions in suitable solvents, preferably isotonic with the blood of the intended patient). Parenteral dosage forms may be sterilised (for example by micro-filtration and/or using suitable sterilising agents [such as ethylene oxide]).

Optionally one or more of the following pharmaceutically acceptable adjuvants suitable for parenteral administration may be added to parenteral dosage forms: local anaesthetics, preservatives, buffering agents and/or mixtures thereof.

Parenteral dosage forms may be stored in suitable sterile sealed containers (for example ampoules and/or vials) until use. To enhance stability during storage the parenteral dosage form may be frozen after filling the container and fluid (for example water) may be removed under reduced pressure.

Pharmaceutical compositions and formulations may be administered nasally in known pharmaceutical forms for such administration (for example sprays, aerosols, nebulised solutions and/or powders). Metered dose systems known to those skilled in the art (for example aerosols and/or inhalers) may be used.

Pharmaceutical compositions and formulations may be administered to the buccal cavity (for example sub-lingually) in known pharmaceutical forms for such administration (for example slow dissolving tablets, chewing gums, lozenges, pastilles, gels, pastes, mouthwashes, rinses and/or powders).

Compositions and formulations for topical administration may comprise a matrix in which the pharmacologically active compounds of the present invention are dispersed so that the compounds are held in contact with the skin in order to administer the compounds transdermally. A suitable transdermal composition may be prepared by mixing the pharmaceutically active compound with a topical vehicle, such as a mineral oil, petrolatum and/or a wax, for example paraffin wax or beeswax, together with a potential transdermal accelerant such as dimethyl sulphoxide or propylene glycol. Alternatively the active compounds may be dispersed in a pharmaceutically acceptable cream or ointment base. The amount of active compound contained in a topical formulation should be such that a therapeutically effective amount of the compound is delivered during the period of time for which the topical formulation is intended to be on the skin.

In the compositions of the present invention, the protein and/ or the microparticles or microcapsules may, if desired, be associated with other compatible pharmacologically active ingredients.

Agrochemical compositions of the invention are compositions that are intended for application to growing plants, or areas where plants (especially food crops) are grown, in order to benefit the health and/or growth of the plant, either directly (for example, by acting on the plant) or indirectly (for example, by the control of insects). Agrochemical compositions may be in the form of solids (e. g. , powders), semi-solids<BR> (e. g. , gels or pastes) or liquids (e. g. , emulsions, suspensions, dispersions or solutions). The compositions are preferably in the form of sprayable liquids, or liquid concentrates which are capable of being sprayed when mixed with water. The compositions preferably comprise a wetting agent, such as a surfactant, and may also comprise one or more other emulsifying agents.

Micro-particles and Micro-capsules The microparticles and microcapsules for use in the present invention may be produced by methods known in the art.

Microcapsules and microparticles useful in the invention preferably consist of populations in which at least 90% (more preferably at least 95%) of the particles have an average particle size (based on the maximum dimension of the particles, such as the diameter when they are spherical) of from 0.05 pm to 2.0 mm, more preferably from 0.5 um to 200 um, most preferably from 10 um to 80 um. Particle sizes can be

determined by microscopy using standard graticule slides, as is well known in the art.

Micro-encapsulation is a generic term used to describe a variety of micro-particulate constructs that include micro- capsules, micro-spheres, nano-particles, nano-capsules and liposomes. These constructs may be spherical, oblong, irregularly shaped, monolithic or aggregates comprising single or multiple walls. Within this diverse group of micro- encapsulate descriptors two sub-categories of particle constructs are readily differentiated: those with a discrete continuous outer coat, film or membrane where the core is mainly concentrated near the centre and termed reservoir design; and those in which the core is substantially dispersed throughout the matrix and called matrix design.

In pharmaceutical and biomedical applications these constructs are frequently termed either micro-capsules or micro-particles, respectively; the same terminology is used herein.

A variety of terms are used to describe the contents of micro- capsules including core material, core, substrate, ingredient, and active agent whereas the coating material is termed shell, coat, wall, encapsulating matrix, encapsulating agent and carrier.

A number of different techniques can be used to form micro- capsules or microparticles for use in the present invention.

In general, methods of producing micro-capsules fall into three distinct categories:

Physico-chemical processes, including simple or complex coacervation (aqueous phase separation), emulsion solvent evaporation (organic phase separation), emulsion-solidification and liposome entrapment.

Chemical processes, including interfacial polymerisation and molecular inclusion.

Physical processes such as spray-drying, spray-coating, spray- granulation, prilling and extrusion.

The coating or shell material used for micro-encapsulation purposes include many natural and synthetic polymers typically forming between 1% and 70% of the micro-capsules by weight.

Coating materials useful in the production of micro-capsules include: gums, for example gum arabic, agar, sodium alginate and carrageenan; carbohydrates, for example starch, dextran, sucrose and corn syrup; celluloses, for example carboxymethylcellulose, methylcellulose, ethylcellulose, nitrocellulose, acetylcellulose, cellulose acetate-phthalate and cellulose acetate-butylate-phthalate ; lipids, for example wax, paraffin, tristearin, stearic acid, monoglycerides, diglycerides, beeswax, oils, fats and hardened oils; inorganics, for example calcium sulphate, silicates and clays; and proteins, for example, gluten, casein, gelatine and albumin.

Coating materials are selected on the basis of the chemical and physical chemical properties of the core material, the micro- encapsulation process to be used and the desired properties of the micro-capsules such as release characteristics. For example, water-soluble polymers are used to micro-encapsulate poorly soluble core materials, whereas water insoluble polymers

are used with water soluble core materials. The overall performance of the coating polymers can be modified by altering coat thickness and the use of plastisizers, cross-linkers or multiple coatings, however in general the coating material should not either solubilise or react with the core material.

Examples of other polymers that may be used include those used in the preparation of bio-degradable micro-spheres such as poly (amides), poly (amino acids), poly (alkyl-a-cyano acrylates), poly (esters), poly (orthoesters), poly (urethanes) and poly (acrylamides). Most frequently studied however are the thermoplastic poly (esters) polylactide (PLA), polyglycolide (PGA) and in particular poly (lactide-co-glycolide) (PLGA).

The physical micro-encapsulation process of spray-drying is the most commonly used technique of micro-encapsulation. Spray- drying is defined as the transformation of a fluid state starting material (solution, dispersion or paste) into a dried particulate form via the process of spraying into a hot drying medium. The micro-capsules formed by spray-drying fall within the matrix type design category with the contents existing as micro-particles or micro-droplets distributed within a dry solid support matrix. Although often considered a dehydration process micro-capsules are formed when active materials become trapped within the matrix formed from a polymer or melt.

Despite a size distribution determined by many process variables particles formed using the spray-drying process are typically under 100pm in size.

Utility

The compositions of the invention may be used in the pharmaceutical or healthcare fields. The compositions can be used to deliver a molecule or substance that is either <BR> <BR> biologically active (e. g. , a pharmaceutical or an antibacterial or antifungal agent) or otherwise therapeutically useful for the treatment or prevention of disease or the alleviation or prevention of pain or discomfort. Specific examples of applications of the compositions are their use as delivery vehicles (e. g. , to deliver biologically active molecules or substances to a desired site). Other medical applications include the manufacture or treatment of sterile articles for use in medicine such as bandages, dressings, medical devices and fabrics used in medicine. Examples include wound dressings (e. g. , bandages, plasters and burn dressings), cotton buds,<BR> supports (e. g. , for joints such as the knee, elbow, wrist or neck), cotton wool, wipes and tissues, and other specialist dressings. The proteins may be used to deliver benefit agents such as antibacterial or antifungal agents to these articles.

Other examples of medical applications include use in spray on skin, self-disinfecting surgical garments (including clothes, drapes and masks), swabs, dental articles and equipment (including toothbrushes, toothpicks and dental floss), surgical tape, brushes and combs, garments including socks for athletes foot or verucas, gloves or other garments for the treatment of eczema, hair accessories and hats, skin/nail and hair treatments, capsules, prosthetics and medical implants (e. g., fibreglass as used in casts for broken limbs), liners to fit between prosthetic limbs and their attachment point, chewing gum, toothpaste, medicated shampoos, and targeted sunscreen for garments (skin protection).

In this and other embodiments of the invention, the proteins have the advantage that they. can be recharged with the benefit agent. Thus, a single treatment with the protein can be followed by multiple treatments with the benefit agent which then has increased affinity for the article as a result of binding via the protein that can remain bound to the surface of the article.

In another embodiment, the compositions of the invention may be used in the agrochemicals field. The compositions can be used to deliver materials that are beneficial for plants either directly, for example as nutrients or selective weedkillers, by killing or repelling plant pests or other pathogens (such as fungi) or indirectly by attracting beneficial insects or animals. Specific examples of applications include: the treatment of plant cell walls; killing or repulsion of plant pests; the attraction of beneficial animals and insects; the treatment of bark, labels, fence posts, decking, tree protectors or tape; the production of a nutrient-releasing, fungicidal or insecticidal article for insertion into the ground (e. g. , a peg); targeting roots for destruction (i. e., getting rid of remaining tree roots); destruction of infestations; slow or delayed release of molecules for insecticidal action; mole deterent e. g. , by release of the Imperial lily odour ; cat deterrent; targeting nutrients to seeds; and bird repellent.

The compositions of the invention in another embodiment can be used in the paper treatment, manufacture or processing e. g. , on a large scale (greater than 100 kg of paper treated, manufactured or processed per day) in the paper industry. The invention therefore contemplates a method of treating paper that comprises applying to the paper a protein which comprises

a first binding domain which is a carbohydrate binding domain and a second binding domain capable of binding to (a) a microcapsule or micro-particle, wherein the microcapsule or microparticle comprises a material that is beneficial in the processing of paper or in the final paper product or (b) directly to said material. The protein may be used with or without the microcapsule or microparticle, but is used with the material itself when no microcapsule or microparticle is present. The compositions can be used to deliver materials that are beneficial in the processing of the paper or which are beneficial in the final paper product, including, for example, antibacterial agents, antifungal agents, perfume or fragrance, flavourants, surfactants, insect repellents, moisturising agents, organic solvents, binding agents for the paper and adhesives. Examples of uses in the paper industry include: wallcovering (e. g. , wallpaper and border) manufacture and treatment, where the compositions of the invention may be used as cleaners, for aroma release, for colour reactivity, for stain removal or for coating ; in the production of specialty paper products such as anti-odour shoe liners, moth repellents, tissues with additives or extra benefits, packaging paper having controlled release of fungicides, packaging that will disintegrate easily when required, food wrappers that have labels impregnated with food aroma; in the manufacture or printing of newspaper ; to release molecules that are used to simplify the paper production process or allow strengthening e. g. , strengthened paper for furniture production or kitchen roll; to produce scented books; for the restoration or preservation of books or manuscripts; in paper diagnostics (e. g. , for fitting to urinals); in the application of an active material to toilet tissue ; as a paper money cleaner or sanitiser; in the production of washable cards (such as identity cards or driving licences); impregnated wipes for

cleaning spectacles or car windscreens ; cloths for the targeted deposition of actives to kitchen surfaces ; in the treatment of greaseproof paper to make it non-stick ; in the formation of labels for packaging (such as cartons, bottles and cans, etc) that will disintegrate within the shelf life of the product; and in the production of paper comprising washing powder and/or fabric conditioner.

Other applications include the treatment of non-paper cellulosic articles. Therefore, the invention also envisages a method of treating a cellulosic substrate with a protein of the invention. Examples include treatments for wooden curtain poles e. g. , for perfume release ;. dust repellents e. g. , for spraying on lampshades ; carpet fresheners ; sprays for refreshing paper drawer liners ; for the urine-triggered release of perfume or odour neutraliser ; to form soft furniture binders; and the treatment of wooden items such as bread bins and chopping boards. The compositions of the invention may also be used in the veterinary field, for example as a treatment for pet litter or in the production of flea collars for pets.

Some specific and preferred applications of the proteins that are the subject of the present invention are as follows: a. Carbohydrate Anchor Therapeutic System As a first typical pharmaceutical application, a protein is provided which comprises a cellulose binding domain (as a typical example of a carbohydrate binding domain) available for surface binding, and an anti-body domain recognising gelatine micro-capsules containing a therapeutic agent. The pharmaceutical application for this construct is useful, for

example in dressings, wound care products and medical devices for internal or external use comprising cellulose or another carbohydrate. A suitable dressing is, for example, a cotton dressing containing a bound CBD construct where the micro- capsule contains an anti-microbial agent or antibiotic to be released through the action of microbial proteolytic enzymes. A medical device that benefits from this invention includes, for example, a urinary catheter designed for long term use without the risk of frequent urinary tract infections. A therapeutic advantage in both examples is the release of anti-microbial agents only in the event of significant infection thereby reducing the spread of anti-biotic resistance among normal bacterial populations. Examples of drugs include antibiotics of the penicillin type e. g. ampicillin, amoxycillin, aziocillin, benzylpenicillin, cloxacillin and also many cephalosporins.

Other antibiotics include but are not restricted to doxycyline, tetracycline, cefaperazone, chloramphenicol and cefoxitin. The carbohydrate binding domain may be used in this approach to bind at a therapeutically useful binding site. For example a therapeutically useful binding site that may be recognised by the carbohydrate binding domain is the HIV envelope glycoprotein expressed on the surface of virus particles and also by infected cells and may therefore provide the means to more effectively target HIV drugs. b. Biphasic Delivery System A construct is provided containing a cellulose binding domain (as a typical example for a carbohydrate binding domain) bound to a micro-particle or micro-capsule comprising cellulose and at least one therapeutic agent together with an anti-body domain recognising gelatine micro-capsules containing at least one therapeutic agent. An advantage of this construct is the

ready formation of a depot of at least one therapeutic agent for biphasic release. This is particularly important for the delivery of certain hormones such as insulin for the treatment of diabetes. Insulin is normally released in a biphasic manner from the pancreas following the ingestion of food, an initial rapid release of insulin is followed by sustained slow release of insulin. This insulin release profile is important in maintaining normal glucose homeostasis but is difficult to achieve using conventional formulation techniques. In the present invention the biphasic release of insulin is controlled by the composition of the two separate insulin-containing micro-capsules or bound to either the carbohydrate binding domain or an anti-body binding domain recognising gelatine micro-capsules formulated for rapid or sustained release.

Insulin-containing proteins of this type are suitable either for depot injection, oral delivery (providing they are protected from the normal digestive processes) or by inhalation enabling biphasic delivery of insulin to be achieved from the lungs. An advantage of insulin delivery via the oral or pulmonary route is the elimination or reduced need for daily insulin injections or perhaps the more effective treatment of type 2 diabetics normally treated with oral hypoglycaemic agents. c. Site Specific Delivery System A protein suitable for site-specific delivery uses the cellulose binding domain (as typical example of a carbohydrate binding domain) to bind a carbohydrate-containing micro-capsule or micro-particulate containing at least one therapeutic agent, whereas another portion of the protein comprises the site- specific targeting moiety. This targeting moiety is preferably in the form of an anti-body recognising a specific disease site

or alternatively a receptor expressed by cells of tissue at or near the site where the therapeutic agent is most required. An example of a suitable application is the delivery of an anti- cancer drug to a tumour site. More specifically, this may involve the use of a protein with an anti-body region recognising prostate specific antigen expressed by prostate tumour cells and a micro-particle or micro-capsule bound to the carbohydrate binding domain containing anti-cancer drug that would therefore be concentrated at the disease site. This will have the effect of both increasing therapeutic efficacy by forming a higher drug concentration at the tumour site while reducing the overall dose for the same therapeutic effect, thereby reducing the likelihood and severity of side effects.

Examples of anti-cancer drugs include alkylating agents, chlorabucil, melphalan, busalfan, lomustine, carmustine, estramustine and thiotepa ; cytotoxic antibiotics, doxorubicin, epirubicin, daunorubicin, mitoxantrone, bleomycin and mytomycin; anti-metabolites, methotrexate, cytarabine, fludarabine, cladribine, gemictabine, fluorouracil ; vinca alkaloids, vinblastine, vincristine, vindesine, vinorelbine, together with other anti-neoplastic agents such as amsacrine, crisantapase, dacarbazine, temozolomide, pentostatin, carboplatin, cisplatin, oxaliplatin, paclitaxel and docetaxel. d. Site Specific Biphasic Delivery System (Multi-binder system) The protein which is provided here combines the advantages of both site-specfic and biphasic delivery using an anti-body construct that has at least three binding sites one of which is a carbohydrate binding site. The carbohydrate binding site is used to bind, e. g. , a micro-particle or micro-capsule containing at least one therapeutic agent with the release rate

being determined by the composition of the micro-capsule or micro-particle. In addition, at least one binding domain is used to bind an additional micro-capsule containing at least one therapeutic agent with the release rate being determined by the composition of the micro-capsule. An example of a therapeutic application for the site specific biphasic delivery system is the enhanced treatment of Parkinson's disease.

Parkinson's disease which is characterised by a progressive loss of motor control is treated with the drug levodopa, a precursor for the neuro-transmitter dopamine in the brain.

Unfortunately, only small amounts of levodopa are absorbed across the blood-brain barrier and as a result levodopa is available for conversion into dopamine by peripheral tissues causing cardiovascular side-effects, nausea and vomiting. To reduce the conversion of levodopa by peripheral tissues a dopa- carboxylase inhibitor is used such as carbidopa. The present invention is contemplated to improve the treatment of Parkinson's disease in the following way: The protein would contain an antibody recognising a binding site expressed on the blood brain barrier such p-glycoprotein. Once bound to the blood-brain barrier a bolus release of dopa-carboxylase inhibitor would be released from one of the bound micro- capsules to reduce peripheral dopamine production while a sustained release of levodopa would occur from another bound micro-capsule. The close proximity to the blood-brain barrier of levodopa release would facilitate absorption into the brain with the effect of either increasing therapeutic efficacy or reducing the dose required for controlling symptoms.

An alternative application of the site-specific biphasic delivery system is to enhance cancer chemotherapy as described in a previous example above. Instead of just relying on one bound micro-capsule or micro-particle containing at least one

therapeutic agent two or more could be directed to the tumour site. For example, a protein comprising a carbohydrate binding domain bound to a carbohydrate-containing micro-capsule containing at least one anti-cancer agent, another binding domain bound to a gelatine micro-capsule containing at least one alternative anti-cancer agent and a binding domain recognising the tumour site or tissue close to the tumour site.

A particular advantage of this arrangement is the ability to combine the site-specific delivery of at least two different anti-tumour agents of widely different physical chemistry. For example, the carbohydrate containing micro-capsule or. micro- particle may contain at least one relatively water soluble therapeutic agent whereas the gelatine micro-capsule may be used to contain at least one oil soluble therapeutic agent. A particular advantage of this combined site specific delivery is overcoming drug resistance of certain tumours resulting from prior chemotherapy.