Field of the invention

The invention relates to novel proteins, and it also relates to a process for the production of such proteins and to the use of such proteins in the production of medicines .

Background of the invention

The complement system is one of the most important

components of the innate immunity of human and animal organisms. The complement system, as the immune system in general, is able to recognise, label and remove intruding pathogens and altered host structures (e.g. apoptotic cells) . The complement system, as a part of the innate immune system, forms one of the first defence lines of the organism against pathogenic microorganisms, but it also links to the adaptive (acquired) immune system at several points forming a bridge, as it were, between innate and adaptive immune mechanism (Walport 2001a; Walport 2001b; Morgan 2005). The complement system is a network consisting of about 30 protein components, which components can be found in the blood plasma in soluble form, and also in the form of receptors and modulators (e.g. inhibitors) attached to the surface of cells. The main components of the system are serine protease zymogens, which activate each other in a cascade-like manner in strictly determined order. Certain substrates of the activated proteases are proteins

containing a thioester bond (components C4 and C3 in the complement system) . When these substrates are cleaved by the activated proteases, the reactive thioester group becomes exposed on the surface of the molecule, and in this way it is able to attach the cleaved molecule to the surface of the attacked cell. As a result of this, such cells are labelled so that they can be recognised by the immune system.

The biological functions of the complement system are extremely diverse and complex, and up till now they have not been explored in every detail. One of the most

important functions is direct cytotoxic activity, which is triggered by the membrane attack complex (MAC) formed from the terminal components of the complement system. The MAP perforates the membrane of cells recognised as foreign, which results in the lysis and, thereby, destruction of such cells.

Another important function of the complement system is opsonisation, when the active complement components (e.g. Clq, MBL, C4b, C3b) settling on the surface of the cells promote the phagocytosis by leukocytes (e.g. macrophages). These leukocytes engulf the cells to be destroyed.

Furthermore, the inflammation initiation role of the complement system is also of outstanding importance. The cleavage products released during complement activation initiate an inflammatory process through their chemotactic stimulating effects on leukocytes (Mollnes 2002).

The components of the complement system are present in blood plasma in an inactive (zymogenic) form until the activation of the complement cascade is triggered by an appropriate signal (e.g. intrusion of a foreign cell, pathogen) . The normal activity of the complement system is important from the aspect of maintaining immune

homeostasis. Both its abnormal underactivity and its uncontrolled hyperactivity may result in the development of severe diseases or in the aggravation of already existing diseases (Szebeni 2004) .

The complement system can be activated via three different pathways: the classical pathway, the lectin pathway and the alternative pathway. In the first step of the classical pathway the CI complex binds to the surface of the

activator, that is the biological structure recognised as foreign. The CI complex is a supramolecular complex

consisting of a recognition protein molecule (Clq) and serine proteases (Clr, Cls) associated to it (Arlaud 2002) . First of all the Clq molecule binds to immune complexes, apoptotic cells, C-reactive protein and to other activator structures. As a result of the Clq molecule binding to the activator, the serine protease zymogens present in the CI complex become gradually activated. In the tetramer Cls- Clr-Clr-Cls first the Clr zymogens autoactivate, then the active Clr molecules cleave and activate the Cls molecules. The active Cls cleaves the C4 and C2 components of the complement system, which cleavage products are the

precursors of the C3-convertase enzyme complex (C4bC2a) . The C3-convertase splits C3 components and transforms into C5-convertase (C4bC2aC3b) . The C5-convertase cleaves C5, after which the activation of the complement system

culminates in the terminal phase characteristic of all three pathways (formation of the MAC) . The activation of a different pathway of the complement system, the lectin pathway, is very similar to that of the classical pathway (Fujita 2004). However, in this case several different types of recognition molecules are involved: MBL ( "mannose-binding lectin") and ficolins (H, L and M types). These molecules bind to the carbohydrate structures on the surface of microorganisms. The binding of the recognition molecule is followed by the autoactivation of MASP-2 ("MBL-associated serine protease"-2) zymogen. The activated MASP-2 cleaves the C4 and C2 components, which results in the formation of the C3-convertase enzyme complex already described in the course of the classical pathway, and from this point the process continues as described above.

The alternative pathway starts with the cleavage of the C3 component and its anchoring to the surface of the

biological structure recognised as foreign (Harboe 2008). If the C3b component created during the cleavage is bound to the cell membrane of a microorganism, then at the same time it also binds the zymogenic form of a serine protease called factor B (C3bB) , which is activated by factor D present in the blood in active form, by cleavage. The C3bBb complex created in this way is the C3-convertase of the alternative pathway, which, after being completed with a further C3b molecule, transforms into C5 convertase. The alternative pathway may also be triggered spontaneously, independently, by the slow hydrolysis of the C3 component (C3w) , but if either the classical or the lectin pathway gets to the point of C3 cleavage, the alternative pathway significantly amplifies their effect. Of the pathways above, we describe the lectin pathway in greater detail, which has been recently discovered and has been characterized the least, and which is the most

important from the aspect of the present invention. Several different types of proteases and non-catalytic proteins bind to the recognition molecules present in several different forms (MBL of different degrees of polymerisation and ficolins) . MASP-2 even in itself is able to initiate the complement cascade (Ambrus 2003; Gal 2005) , but this latter enzyme is present in a smaller amount (0.5 μg/ml) than MASP-1. The physiological function of the MASP-1 protease present in a higher amount (7 g/ml) has not been completely explored yet. Although MASP-1 on its own is not able to initiate the complement cascade (it can only cleave C2 but not C4), its activity may supplement the activity of MASP-2 at several points, therefore active MASP-1 may be necessary for amplifying and consummating the effect of the lectin pathway. Several signs indicate that to a certain extent

MASP-1 is a protease similar to thrombin, forming a bridge between the two major proteolytic cascade systems - the complement system and the blood coagulation system - in the blood (Hajela 2002; Krarup 2008) .

The gene of both MASP-1 and MASP-2 has an alternative splicing product. The MApl9 (sMAP) protein is produced from the MASP-2 gene, containing the first two domains of MASP-2 (CUB1-EGF) . The Map44 and the MASP-3 mRNA are transcribed from the MASP-1 gene. Similarly to the MApl9, the MAp44 is also a truncated protein: it contains only the first four non-catalytic domains CUB1-EGF-CUB2-CCP1 ) , so it does not have proteolytic activity (Degn 2009) . Its function is unknown, it probably plays a role in regulation. The first five domains of MASP-3 are the same as the domains of MASP- 1, but they differ in their serine protease domain. MASP-3 has low proteolytic activity on synthetic substrates, and its natural substrate is not known. Unlike other early proteases, it does not form a complex with the Cl-inhibitor molecule. Probably the presence of MApl9, Map44 and MASP-3 acts against the activation of the lectin pathway, as these proteolytically inactive proteins compete with the active MASP-2 and MASP-1 enzymes for the binding sites on the recognition molecules.

As it has been mentioned above, abnormal operation of the complement system in the human or animal organism may result in developing disease. The uncontrolled activation of the complement system may result in damaging self- tissues, and developing inflammatory or autoimmune

conditions (Beinrohr 2008) . One of these conditions is ischemia-reperfusion (hereinafter: IR) injury, which occurs, when the oxygen supply of a tissue is temporarily restricted or interrupted (ischemia) for any reason (e.g. vascular obstruction) , and after the restoration of blood circulation (reperfusion) cellular destruction starts.

During reperfusion the complement system recognises

ischemic cells as altered self cells and starts an

inflammatory reaction to remove them. Partly this

phenomenon is responsible for tissue damage occurring after cardiac infarction and stroke, and it may also cause complications during coronary bypass surgery and organ transplantations (Markiewski 2007) . The lectin pathway probably plays a role in the development of IR injury. For this reason the deliberate suppression of the lectin pathway may reduce the extent and the consequences of IR injury. The lectin pathway may also become activated in the case of rheumatoid arthritis (hereinafter: RA) as MBL binds to the antibody form IgG-GO having altered glycosylation accumulated in the joints during RA. The uncontrolled activity of the complement system also plays a role in the development and maintenance of different neurodegenerative diseases (e.g. Alzheimer's, Huntington's and Parkinson's diseases, Sclerosis Multiplex) , and it is one of the main factors in the pathogenesis of age-related macular

degeneration (AMD) as well (Bora 2008) . The latter clinical picture is responsible for half of all cases of age-related loss of eyesight in developed industrial countries. The complement system can also be associated with one of the forms of autoimmune nephritis (glomerulonephritis) and with another autoimmune disease, namely SLE (systemic lupus erythematosus) . The intravenous administration of certain diagnostic agents, especially when they are used in

liposomes, may generate allergic type reactions independent from antibodies in patients. This pseudo-allergic reaction is due to the activation of the complement system

(complement activation-related pseudo-allergy, abbreviated as CARPA) . The released inflammation initiating substances

(e.g. C5a, C3a) mobilise the cellular elements of the immune system. With the selective inhibition of the

complement system fatal pseudo-allergic reactions can be suppressed .

If the complement system is inhibited during the first steps, the efficient and selective inhibition of certain activation pathways becomes possible without triggering general immunosuppression. By inhibiting MASP-1 and MASP-2 enzymes the lectin pathway can be blocked selectively (e.g. in the case of the diseases mentioned above) , and by this the classical pathway responsible for the elimination of immunocomplexes is left untouched, that is functioning.

The Clr, Cls, MASP-1, MASP-2 and MASP-3 enzymes form an enzyme family having the same domain structure (Gal 2007) . The trypsin-like serine protease (SP) domain responsible for proteolytic activity is preceded by five non-catalytic domains. The three domains CUB1-EGF-CUB2 forming the N- terminal part of the molecules (CUB = Clr/Cls, sea urchin Uegf and Bone morphogenetic protein-1; EGF = Epidermal

Growth Factor) are responsible for the dimerization of the molecules (both in the case of MASP-1 and MASP-2) and for interacting with other molecules, e.g. for binding to the recognition molecules.

The C-terminal CCP1-CCP2-SP fragment (CCP = Complement Control Protein) of the molecules is equivalent to the whole of the molecule in respect of its catalytic

properties. One of the characteristic features of

complement proteases is that they have very narrow

substrate specificity, they are able to cleave the well- defined peptide bonds of only a few protein substrates. Both the CCP modules and the SP domain contribute to this finely tuned specificity.

The SP domain contains the active centre characteristic of serine proteases, the substrate binding pocket and the oxyanion hole. Eight surface loop regions, the conformation of which is quite different in the different proteases, play a decisive role in determining subsite specificity.

On the one part the CCP modules stabilise the structure of the catalytic region, and on the other part they contain binding sites for large protein substrates. Although the small-molecule compounds generally used for inhibiting trypsin-like serine proteases (e.g. benzamidine, NPGB, FUT- 175) inhibit the activity of complement proteases too

(Schwertz 2008), this inhibition is not selective enough; it also extends to the inactivation of other serine

proteases in the blood plasma, e.g. blood coagulation enzymes, kallikreins. The only known natural inhibitor of the complement system, CI inhibitor protein circulating in blood and belonging to the serpin family is also characterised by relatively wide specificity . The first lectin pathway selective inhibitors according to the prior art, developed by the inventors of the present invention were described in Hungarian patent application no. P0900319. The reversibly binding peptide inhibitors according to this earlier invention inhibit the activation of the lectin pathway by inactivating the MASP-1 and MASP-2 enzymes, while the classical and the alternative pathways remain intact. These earlier inhibitors are based on the SFTI (Sunflower Trypsin Inhibitor) , and in a functional sense two types have been developed: the inhibitor

inhibiting the MASP-2 enzyme with exclusive selectivity, which does not inhibit the MASP-1 enzyme, and another inhibitor, which inhibits both proteases but is able to inhibit the MASP-1 enzyme about fifteen times more

efficiently than the MASP-2 enzyme.

Summary of the invention

The inhibition of the complement system, including the lectin pathway, may be an efficient tool in fighting against human and animal diseases occurring as a result of the abnormal activity of the complement system. The

presently known inhibitors having the SFTI structure can either inhibit both proteases (MASP-1 and MASP-2) or they are selective MASP-2 inhibitors.

For this reason we set the aim to develop compounds, which are more efficient than the known SFTI-based inhibitors and have a greater selectivity.

The proteins according to the present invention show a greater affinity to the target enzymes and are more

selective with respect to the MASP-1 and MASP-2 enzyme than the known inhibitors according to the prior art.

One of the main aims of increasing specificity is to throw light upon the specific role of the individual MASP enzymes in the lectin pathway and to open up more selective

therapeutic possibilities by this. Surprisingly we found that the following proteins

containing sequences according to general formula (I) are suitable for the above objectives:

X1CTX2X3X4CX5 (i; where

Xi is M, F, V, A, I, and

X ₂ is R, K, and

X ₃ is K, R, L, A and

X ₄ is L, G, M, A, , Y, and

X ₅ is W, Y, L, M, N, E, G.

In accordance with the above, the invention relates to proteins containing sequences according to general formula (I), their salts, esters and pharmaceutically acceptable prodrugs .

More preferably the invention relates to proteins

containing the following sequences:

MCTRKLCW (SEQ ID NO 1),

MCTRKLCY (SEQ ID NO 2),

FCTRKLCY (SEQ ID NO 3),

ACTRKLC (SEQ ID NO 4),

VCTRLWCE (SEQ ID NO 5),

VCTRLWCN (SEQ ID NO 6),

VCTRLYCN (SEQ ID NO 7),

VCTKLWCN (SEQ ID NO 8),

and their salts or esters.

Most preferably the invention relates to proteins

containing the following sequences:

FCTRKLCY (SEQ ID NO 3),

VCTKLWCN (SEQ ID NO 8),

and their salts or esters.

Preferably the proteins according to the invention are modified proteins within the pacifastin family, especially preferably modified SGCI protein, where the modification lies in that the protein contains one of the sequences according to formula (I) .

Furthermore, the invention also relates to pharmaceutical preparations that contain at least one protein containing sequences according to general formula (I), its salt, ester or prodrug and at least one further additive. This additive is preferably a matrix ensuring controlled active agent release . The invention relates especially to pharmaceutical

preparations that contain at least one of the proteins containing the following sequences:

MCTRKLCW (SEQ ID NO 1),

MCTRKLCY (SEQ ID NO 2),

FCTRKLCY (SEQ ID NO 3),

ACTRKLCW (SEQ ID NO 4),

VCTRLWCE (SEQ ID NO 5),

VCTRLWCN (SEQ ID NO 6),

VCTRLYCN (SEQ ID NO 7),

VCTKLWCN (SEQ ID NO 8),

and/or their pharmaceutically acceptable salts and esters. The invention most preferably relates to pharmaceutical preparations that contain at least one of the proteins containing the following sequences:

FCTRKLCY (SEQ ID NO 3),

VCTKLWCN (SEQ ID NO 8),

and/or their pharmaceutically acceptable salts and esters.

The invention also relates to kits containing at least one protein containing sequences according to general formula (I), its salt or ester.

The invention also relates to the screening procedure of compounds potentially inhibiting MASP enzymes, in the course of which a labelled protein according to the invention is added to a solution containing MASP, then the solution containing one or more compounds to be tested is added to it, and the amount of the released labelled protein is measured. In this respect the MASP enzyme is preferably MASP-1 or MASP-2 enzyme. The invention also relates to the use of proteins

containing sequences according to general formula (I) and their pharmaceutically acceptable salts or esters in the production of a pharmaceutical preparation suitable for treating diseases that can be treated by inhibiting the complement system. In accordance with this diseases can be selected preferably from the following group: inflammatory and autoimmune diseases, especially preferably ischemia- reperfusion injury, rheumatoid arthritis, neurodegenerative diseases, age-related macular degeneration,

glomerulonephritis, systemic lupus erythematosus, and pseudo-allergy developing as a consequence of complement activation .

The invention also relates to a procedure for isolating MASP enzymes, in the course of which a carrier with one or more immobilised protein with sequences according to general formula (I) are contacted with a solution

containing a MASP enzyme and the preparation is washed. In this respect the MASP enzyme is preferably MASP-1 or MASP-2 enzyme .

Short description of the drawings In the drawings

figure 1 shows a schematic representation of the phage display method;

figure 2 shows the sequence logo diagrams of the obtained sequences, where

figure 2. a shows the sequence diagram relating to the sequences selected on the MASP-1 enzyme and specific to it; figure 2b shows the sequence diagram relating to the sequences selected on the MASP-2 enzyme and specific to it.

Detailed description of the invention

The present invention relates to proteins and protein derivatives selectively inhibiting ASP-1 and MASP-2 enzymes . The present invention also relates to proteins and protein derivatives which are sequentially analogous to the

described sequences and the biological activity of which is also analogous when compared to the described sequences. A person skilled in the art finds it obvious that certain side chain modifications or amino acid replacements can be performed without altering the biological function of the protein in question. Such modifications may be based on the relative similarity of the amino acid side chains, for example on similarities in size, charge, hydrophobicity, hydrophilicity, etc. The aim of such changes may be to increase the stability of the protein against enzymatic decomposition or to improve certain pharmacokinetic

parameters . The scope of protection of the present invention also includes proteins in which elements ensuring detectability (e.g. fluorescent group, radioactive atom, etc.) are integrated. Furthermore, the scope of protection of the present

invention also includes proteins that contain a few further amino acids at their N-terminal, C-terminal, or both ends, if these further amino acids do not have a significant influence on the biological activity of the original sequence. The aim of such further amino acids positioned the ends may be to facilitate immobilisation, ensure the possibility of linking to other reagents, influence

solubility, absorption and other characteristics.

We used the IUPAC recommendations to mark the amino acid side chains in the given sequences (Nomenclature of -Amino Acids, Recommendations, 1974 - Biochemistry, 14(2), 1975).

The present invention also relates to the pharmaceutically acceptable salts of the proteins containing sequences according to general formula (I) according to the

invention. By this we mean salts, which, during contact with human or animal tissues, do not result in an

unnecessary degree of toxicity, irritation, allergic symptoms or similar phenomena. As non-restrictive examples of acid addition salts the following are mentioned:

acetate, citrate, aspartate, benzoate, benzene sulphonate, butyrate, digluconate, hemisulphate, fumarate,

hydrochloride, hydrobromide, hydroiodide, lactare, maleate, methane sulphonate, oxalate, propionate, succinate,

tartrate, phosphate, glutamate. As non-restrictive examples of base addition salts, salts based on the following are mentioned: alkali metals and alkaline earth metals

(lithium, potassium, sodium, calcium, magnesium,

aluminium) , quaternary ammonium salts, amine cations

(methylamine, ethylamine, diethylamine, etc.).

In respect of the present invention prodrugs are compounds that transform in vivo into a protein according to the present invention. Transformation can take place for example in the blood during enzymatic hydrolysis. The proteins according to the invention can be used in pharmaceutical preparations, where one or more additives are needed to reach the appropriate biological effect. Such preparations may be pharmaceutical preparations combined, for example, with matrices ensuring controlled active agent release, widely known by a person skilled in the art.

Generally matrices ensuring controlled active agent release are polymers that, when entering the appropriate tissue (e.g. blood plasma), decompose, for example in the course of enzymatic or acid-base hydrolysis (e.g. polylactide, polyglycolide) .

In the pharmaceutical preparations according to the

invention other additives known in the state of the art can also be used, such as diluents, fillers, pH regulators, substances promoting dissolution, colouring additives, antioxidants, preservatives, isotonic agents, etc. These additives are known in the state of the art.

Preferably, the pharmaceutical preparations according to the invention can be entered in the organism via parenteral (intravenous, intramuscular, subcutaneous, etc.)

administration. Taking this into consideration, preferable pharmaceutical compositions may be aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, or solid (e.g. powdered) preparations, which can be transformed into one of the above fluids directly before use. In such fluids suitable vehicles, carriers, diluents or solvents may be for example water, ethanol, different polyols (e.g.

glycerol, propylene glycol, polyethylene glycols and similar substances) , carboxymethyl cellulose, different (vegetable) oils, organic esters, and mixtures of all these substances .

The preferable formulations of the pharmaceutical

preparations according to the invention include among others infusions, tablets, powders, granules,

suppositories, injections, syrups, etc.

The administered dose depends on the type of the given disease, the patient's sex, age, weight, and on the

severity of the disease. In the case of oral administration the preferable daily dose may vary for example between 0.01 mg and 1 g, in the case of parenteral administration (e.g. a preparation administered intravenously) the preferable daily dose may vary for example between 0.001 mg and 100 mg in respect of the active agent. A person skilled in the art finds it obvious that the dose to be selected depends very much on the molecular weight of the given protein used. Furthermore, the pharmaceutical preparations can also be used in liposomes or microcapsules known in the state of the art. The proteins according to the invention can also be entered in the target organism by state-of-the-art means of gene therapy.

If in order to reach the desired medical effect, an active agent selectively inhibiting MASP-1 or MASP-2 is needed, then from the proteins containing sequences according to general formula (I) according to the invention the

selective inhibitory proteins should be preferably

selected. For example the sequence according to the

invention selectively inhibiting the MASP-2 enzyme may be the peptide with the sequence VCTRLYCN (SEQ ID NO 7), while the sequence according to the invention selectively

inhibiting the MASP-1 enzyme may be the peptide with the sequence FCTRKLCY (SEQ ID NO 3) . Generally the rest of the protein does not influence selectivity. However, as it is obvious for a person skilled in the art, a given protein may have parts beyond the sequence part according to the invention, which may influence the inhibitory activity shown towards the MASP target enzymes. Practically such undesired effects should be filtered out by performing experiments, and planning these in advance is quite

difficult and uncertain according to the prior art. At the same time it is pointed out here that according to the present invention proteins are not preferred, which, apart from the sequence part according to the invention, also contain parts showing any interaction with MASP enzymes having a negative influence on inhibition.

The proteins according to the invention can be preferably used in different kits, which can be used for measuring or localising different MASP enzymes (either in a way specific to any MASP enzyme, or both to the MASP-1 and MASP-2 enzymes at the same time) . Such use may extend to

competitive and non-competitive tests, radioimmunoassays, bioluminescent and chemiluminescent tests, fluorometric tests, enzyme-linked assays (e.g. ELISA) ,

immunocytochemical assays, etc.

In accordance with the invention, kits are especially preferable, which are suitable for the examination of the potential inhibitors of MASP enzymes, e.g. in competitive binding assays. With the help of such kits a potential inhibitor' s ability of how much it can displace the protein according to the invention from a MASP enzyme can be measured. In order to detect it, the protein according to the invention needs to be labelled in some way (e.g.

incorporating a fluorescent group or radioactive atom) . The kits according to the invention may also contain other solutions, tools and starting substances needed for

preparing solutions and reagents, and instruction manuals.

The proteins containing sequences according to the

invention according to general formula (I) can also be used for screening compounds potentially inhibiting MASP

enzymes. In the course of such a screening procedure a protein containing sequences according to general formula (I) is used in a labelled (fluorescent, radioactive, etc.) form in order to ensure detectability at a later point. The preparation containing such a protein is added to the solution containing MASP enzyme, in the course of which the protein binds to the MASP enzyme. Following the appropriate incubation period, a solution containing the

compound/compounds to be tested is added to the

preparation, which is followed by another incubation period. The compounds binding to the MASP enzyme (if the tested compound binds to the surface of the enzyme partly or completely at the same site as the sequence of the protein according to the invention, or somewhere else, but its binding alters the conformation of the MASP enzyme in such a way that it loses its ability to bind the protein) displace the labelled protein from the MASP molecule to the extent of their inhibiting ability. The concentration of the displaced proteins can be determined using any method suitable for detecting the (e.g. fluorescent or

radioactive) labelling used on the protein molecules. The incubation periods, washing conditions, detection methods and other parameters can be optimised in a way known by th person skilled in the art. The screening procedure

according to the invention can also be used in high- throughput screening (HTS) procedures.

The proteins according to the invention can be used first of all in the medical treatment of diseases, in the case o which the inhibition of the operation of the complement system has preferable effects. Consequently the present invention also relates to the use of proteins in the production of medicaments for the treatment of such diseases. As it has been explained above in detail, such diseases are first of all certain inflammatory and

autoimmune diseases, especially the following diseases: ischemia-reperfusion injury, rheumatoid arthritis,

neurodegenerative diseases (e.g. Alzheimer's, Huntington's and Parkinson's disease, Sclerosis Multiplex), age-related macular degeneration, glomerulonephritis, systemic lupus erythematosus, complement activation-related pseudo- allergy .

The proteins according to the invention can also be used for isolating MASP proteins, by immobilising proteins and making the preparation made in this way come into contact with the solution presumably containing MASP enzyme. If this solution really contains MASP enzyme, it will be anchored via the immobilised protein. This procedure can b suitable both for analytical and preparative purposes. If the geometry of the binding of the given protein on the MASP enzyme is not known, during this procedure a peptide anchored from several directions or even several proteins should be used to ensure appropriate linking. The solution containing the MASP enzyme can be a pure protein solution, an extract purified to different extents, tissue preparation, etc.

According to the present invention, by proteins containing the sequences according to the invention we mean the following. By such protein we mean any amino acid sequence, which consists of the sequence according to general formula (I) at least. However, preferably this sequence is a part of a larger protein to make sure that the two extreme members of the sequence of general formula (I) according to the invention (that is amino acids marked Xi and X ₅ in general formula (I)) are situated at an appropriate

distance from each other. First of all this appropriate distance can be ensured by the appropriate molecular environment, that is by a protein of an appropriate spatial structure. Due to the appropriate distance between these two extreme amino acids, the sequence part according to the invention can assume the appropriate optimal geometry for inhibiting the MASP enzymes. For this reason the distance measured between the alpha carbon atoms of the two extreme amino acids is preferably 20±4 A. A person skilled in the art finds it obvious that the appropriate distance between the two extreme amino acids of the sequence according to the invention can be ensured by inserting it in a larger protein, and also by adding a suitable shorter sequence part, even a few amino acids, and by creating a covalent or ionic bond between them. For example, cysteine side chains can be inserted a few positions before and after the two extreme amino acids mentioned above, and by creating appropriate conditions between these cysteine side chains, a covalent disulfide bridge can be created. It follows from the above that according to the present invention shorter peptides and modified peptides are also regarded as

proteins .

According to the present invention the protein according to the invention is preferably a protein within the pacifastin family, carrying the sequence according to the invention in the given position. According to the invention, especially preferably the protein is an SGPI-2 protein within the pacifastin family, which has the sequence according to the invention at the given position.

The SGPI-2 protein has the following sequence (SEQ ID NO 13) :

EVTCEPGTTFKDKCNTCRCGSDGKSAACTLKACPQ,

where the underlined part indicates the sequence part to be replaced with the sequence part according to the present invention .

So if for example in accordance with the invention we are talking about a protein, where the protein according to the invention is preferably an SGPI-2 protein which preferably contains the FCTRKLCY (SEQ ID NO 3) sequence, then the protein according to the invention has the following amino acid sequence (SEQ ID NO 14) :

EVTCEPGTTFKDKCNTCRCGSDGKSAFCTRKLCYQ,

where the underlined part indicates the sequence part according to the invention. This sequence (SEQ ID NO 14) is to be regarded exclusively as a preferred embodiment for demonstrating the invention, and not as the limitation of the invention.

On the basis of the above it is obvious that our invention relates to proteins, a part of which is formed by the sequence with general formula (I) according to the

invention. A person skilled in the art also finds it obvious that when pacifastin and SGCI is mentioned as a preferred protein within the framework of the present description, we mean pacifastin and SGCI which has the sequence with general formula (I) according to the

invention in the given position. Such proteins can be referred to as modified proteins within the pacifastin family, or as modified SGCI proteins.

Phage display

The proteins according to the invention were developed using the phage display method.

The phage display is suitable for the realisation of directed in vitro evolution, the main steps of the state- of-the-art procedure (Smith 1985) can be seen in figure 1. In the course of this the gene of the protein involved in evolution is linked to a bacteriophage envelope protein gene. In this way, when the bacteriophage is created, a fusion protein is produced which becomes incorporated into the surface of the phage. The phage particle carries the gene of the foreign protein inside, while on its surface it displays the foreign protein. The protein and its gene are physically linked via the phage. For directed protein evolution, we change the codons of the gene coding it, carefully determined by us. Numerous codons can be changed at the same time using combinatorial mutagenesis based on a mixture of synthetic oligonucleotides. The position of the mutations and variability per position is determined at the same time. After creating a DNA library containing several billions of variants and entering it into bacteria, the phage protein library is created. Each phage displays only one type of protein variant and carries only the gene of this variant. The individual variants can be separated from each other using analogue methods to affinity chromatography, on the basis of their ability to bind to a given target molecule chosen by the researcher (and generally linked to the surface) . At the same time, as opposed to simple protein affinity chromatography, phage protein variants selected in this way have two important characteristic features. On the one part they are able to multiply, on the other part they carry the coding gene wrapped in the phage particle.

During evolution, instead of examining individual mutants, in actual fact billions of experiments are performed simultaneously. Binding variants are multiplied, and after several cycles of selection-multiplication a population rich in functional variants is obtained. From this

population individual clones are examined in functional tests, while the protein is still displayed on the phage. The phage protein variants found appropriate during the tests are identified by sequencing the physically linked gene. Besides the individual measurements, through the sequence analysis of an appropriately large number of function-selected clones it is also revealed what amino acid sequences enable fulfilling the function. In this way a database based on real experiments is prepared which makes it possible to elaborate a sequence-function

algorithm. The variants found the best on this basis are also produced as independent proteins, and these are examined in more accurate further tests. We ourselves developed the vectors suitable for phage display from the vectors available in commercial distribution, they will be described later.

Selecting the inhibitor sceleton

When selecting the inhibitor sceleton it was a condition that the marked inhibitor structure should be: canonic, highly efficient on human trypsin and small. A further expectation in connection with the new inhibitor sceleton was that it should have a protease binding loop with the least possible structural constraints. The structure of the member named SGPI-2 in the pacifastin inhibitor family fulfils these requirements: it is canonic, it can inhibit several different types of human serine protease, and with its length of 35 amino acids it can still be regarded small. As compared to the SFTI inhibitor sceleton included in the earlier Hungarian patent application no. P0900319, there are several positions in the inhibition loop, the side chains of which do not participate in creating the internal structure of the molecule, but they interact with the enzyme. It seemed justified that the guided

evolutionary modification of these positions would result in more selective inhibitors of a higher affinity.

Creating a library

As the basic molecule of the library we chose the SGPI-2 molecule ( Schistocerca Gregaria Peptidase Inhibitor-2 ) , which inhibits chymotrypsin and elastase type mammalian and arthropodal enzymes and is a serine-protease inhibitor within the so-called pacifastin family (Merops ID: 119) . It was isolated from the haemolymph of the desert locust, in which it is selected linked with the SGPI-1 inhibitor in a tandem form, and then the two free inhibitor forms are obtained as a result of proteolytic cleavage. Below we refer to SGPI-2 as SGCI (S. Gregaria Chymotrypsin Inhibitor) . With two point mutations SGCI can be changed into a potent trypsin inhibitor. The inhibitors within the family have a triple antiparallel stranded beta sheet structure consisting of about 35 amino acids. They can be characterised with the following consensus sequence:

C _a X9_i2Cb XC _c C _a X2-3GX3- C _c TX3Cb

The six cysteines form bridges between the three beta strands suiting the abcacb pattern. The inhibition loop contacting the protease is situated near the C-terminus (Malik 1999) .

When creating the library our aim was the complete randomisation of the inhibition loop. Generally the loop is determined with positions P4-P4' (in the case of substrates the environment of the cleaved bond within +/- 4 amino acids; nomenclature: Schechter & Berger 1967) . The two cysteines situated in this region, which have key importance in the stabilisation of the inhibitor' s structure, are left intact. In the other 6 positions we allowed the presence of all 20 amino acids.

The initial SGCI inhibitor loop's amino acid sequence: ACTLKACP (SEQ ID NO 9) .

The amino acid sequence of the inhibition loop characteristic of the library is XCXXXXCX,

where X can be any one of the 20 amino acids. The underlined part indicates the so-called Pi group, which generally bears outstanding significance from the aspect of specificity, as it reaches into deep binding pocket of the enzyme responsible for primary selectivity. In order to be able to select high-affinity binding molecules during phage display, it is essential that the binding molecule displayed should be presented in a low copy number per phage, ideally in one single copy (monovalent phage display) . By this seemingly high-affinity binding (avidity) deriving from simultaneous binding to several anchored target molecules can be avoided. In the system used by us, the phage-SGCI library was created through a glycine-serine linker as the N-terminal fusion of the p8 main envelope protein. In connection with the SGCI molecule described in this way we have pointed out earlier that it appears on the phage surface in one single copy (Szenthe 2007) .

Before the N-terminus of the SGCI library, we also inserted a linear epitope tag recognisable by monoclonal antibodies, using an appropriate distance-keeping peptide link. This was the so-called "Flag-tag", which served two purposes. One of these was to be able to demonstrate easily the displaying of the library on the phage surface. The other purpose was to find out, after sequencing the clones obtained as a result of control selection using the antibody against the tag, clones of what sequence are obtained in the lack of the specific target enzymes, that is MASPl and MASP2. The sequence pattern obtained in this way shows the amino acids in the individual randomised positions which ensure the most efficiently the production of the peptide displayed on the phage and its release from the cell. In this way, when comparing the result of the selection performed on the enzymes to this group selected on the antibodies, the typical position-dependent amino acid preferences that can be really attributed to binding to the enzyme and are not the results of some other effect (e.g. more efficient production) can be revealed.

Examples

Below the present invention is described in detail on the basis of examples, which, however, should not be regarded as examples to which the invention is restricted. Through the examples we show how the phage library was created (Example 1), describe phage selection (Example 2) and introduce the results (Example 3) . In example 4 the method of the heterologous expression of the inhibitors is described together with the relating analytical studies.

Example 1; creating the phage library 1.1. Preparation of a Kunkel-template In the first step, from pGP8-Tag-SGCI phagemid a single- stranded Kunkel-template is prepared, in which stop codons were inserted using Kunkel's method (Kunkel 1985) in the positions to be randomised at a later point. The role of the stop codons is to eliminate possible wild-type SGCI backgrounds while creating the DNA library. The mutagenesis used when creating the library is never 100% efficient, some of the created population is of the same sequence as the template. In our case this population does not appear as a displayed peptide, as it contains numerous stop codons.

The DNA library was also created using Kunkel's method. For this we used degenerate oligonucleotides. The DNA library created in this way was introduced into supercompetent cells by electroporation . The phage protein library was created by the helper phage infection of the cell culture. 1.2. Creating the DNA library

1.2.1. Creating stop mutant phagemid by Kunkel mutagenesis

1.2.1.1. Transformation of CJ 236 E. coli strain

1 μΐ (-100 ng) pGP8-Tag-SGCI phagemid

10 μΐ 5x KCM (0.5 M KC1, 0.15 M CaC12, 0.25 M MgC12)

39 μΐ distilled water

The DNA solution was cooled on ice.

We added 50 μΐ CJ236 cells and it was incubated for 20 minutes on ice. Then we left the cells alone for 10 minutes at room temperature, and after adding 400 μΐ LB medium we shook it for 30 minutes at 37 °C. 100 μΐ from this was grown overnight at 37 °C on an LB-agar + ampicillin (100 μg ml) plate.

1.2.1.2. Production and isolation of uracil-containing phage From a separate colony cells were inoculated in 2 ml 2YT/ampicillin (100 μg/ml) , chloramphenicol (5 μg/ml) medium and grown overnight, shaken at 37 °C. On the following day 30 μΐ culture was inoculated in 30 ml medium of the same composition. As soon as the light dispersion of the cell suspension measured at 600 nm (OD600) reached 0.4, it was infected with M13-K07 helper phage allowing 10 phages per coli cell on average. After shaking it for 30 minutes at 37 °C kanamycin was added to the solution in a final concentration of 25 μg/ml. The cells were shaken for 16 more hours at 37 °C. Then the cells were isolated from the culture by centrifugation (10,000 rpm, 10 minutes, 4 °C) , and from the supernatant containing the phages the phages were precipitated in a clean centrifuge tube: adding 1/5 volume PEG/NaCl solution (20% PEG 8000, 2.5 M NaCl). After thoroughly mixing in the precipitation agent, the sample was left alone for 20 minutes at room temperature. Then the phage particles were settled by centrifuging (12,000 rpm, 10 minutes, 4 °C) . After pouring off the supernatant carefully and putting back the tube in the same position, the liquid stuck to the wall of the tube was collected by centrifuging it for a while (1,000 rpm, 1 minute, 4 °C) and then it was removed with a pipette. The phages were suspended in 800 μΐ PBS, and the remaining cell fragments were removed from the sample by centrifuging it in a microcentrifuge (12,000 rpm, 10 minutes, 4 °C) . The supernatant obtained in this way contained pure phages.

1.2.1.3. Isolation of single-stranded DNA from phages

From the nearly 800 μΐ phage, with the help of a QIAgen Spin M13 kit single-stranded DNA (ssDNA) was isolated following the manufacturer's instructions. The amount of the pure ssDNA was determined on the basis of UV light absorption at 260 nm.

1.2.1.4. Kunkel mutagenesis

Stop mutations were introduced with the following oligonucleotide (SEQ ID NO 10) : 5' -

GCGGTAGCGATGGCAAAAGCGCGTAATGCTAATAATAATAATGCTAACAGGGTACCG G TGGAGG-3' 1.2.1.4.1. Oligo phosphorylation 2 μΐ oligo (330 ng/μΐ)

2 μΐ 10X TM buffer (0.5 M Tris-HCl pH 7.5, 0.1 M MgC12) 2 μΐ 10 mM ATP

1 μΐ 100 mM DTT

12 μΐ distilled water

1 μΐ polynucleotide kinase (NEB, lOU/μΙ)

The reaction was incubated for 30 minutes at 37 °C. 1.2.1.4.2. Oligo-template annulation

1 μg ssDNA

2 μΐ from the kinase oligo reaction mixture

2,5 μΐ 10X TM buffer

Distilled water up to a final volume of 25 μΐ

Incubation: 90 °C 1 minutes, 50 °C 3 minutes, then after centrifuging it for a while it was put on ice.

1.2.1.4.3. Polymerisation and ligation

The following were added to the above DNA solution:

1 μΐ 10 mM ATP

1 μΐ 25 mM dNTP

1.5 μΐ 100 mM DTT

0.6 μΐ T4 ligase (NEB, 400ϋ7μ1)

0.6 μΐ T7 polymerase (NEB, lOU/μΙ)

The reaction was incubated for 2 hours at 37 °C. The whole mixture was run on 0.8% agarose gel, the product of the desired size was cut out from the gel. From this piece of gel, with the help of a QIAgen Gel Extraction kit the Kunkel product was isolated in 30 μΐ elution buffer (EB) . With the Kunkel product XLl Blue cells were transformed, using 10 μΐ DNA as described above. From individual colonies cell cultures were grown in LB/ampicillin (100 μς/πιΐ) medium. From the cells the phagemid was isolated with a QIAgen Spin Miniprep kit, following the manufacturer's instructions. The DNA construction was checked with sequencing (ABI PRISM BigDye v3.0 Kit). The name of the vector created in this way: pGP8-Tag-SGCI-STOP phagemid. 1.3. Library Kunkel mutagenesis

The library mutagenesis was realised in a similar way as described above in point 1.2., but using ten times the amounts determined therein. The library oligo is analogous with the stop mutation oligo, but here there are degenerate triplets on the place of the TAA stop codons . The library oligo sequence, using the IUPAC coding relating to degenerate oligonucleotides, was the following (SEQ ID NO

11) :

5' -

GCGGTAGCGATGGCAAAAGCGCGNNKTGCNNKNNKNNKNNKTGCNNKCAGGGTACCG G TGGAGG-3'

Oligo phosphorylation was performed as described above. Here, for the library oligo template annulation ten times the amount of the oligo was used, so all the oligo created during the kinase reaction was used. The template for the mutagenesis was the uracil-containing ssDNA carrying the stop codons, which was created from the pGP8-Tag-SGCI-STOP phagemid obtained as a result of the procedure described above in detail, in CJ236 cells, by M13K07 helper phage infection. For creating the library ten times the amount of the template was used: 20 μg, and the annulation volume was also increased by ten times to 250 μΐ . The incubation periods were extended: 90°C 2 minutes, 50°C 5 minutes. The amounts were multiplied by ten for polymerisation too, the reaction took place overnight at 16 °C.

The product was cleaned with a Qiagen Gel Extraction kit, it was not isolated from gel, only cleaned on the column. Elution took place in 2 x 60 μΐ USP distilled water. 1.4. Electroporation, multiplication of the phage library

The library was introduced to the supercompetent cells via electroporation. Our aim was to introduce the plasmid to as many cells as possible, so that our library contains 10 ⁸ -10 ⁹ pieces.

The DNA library, which is situated in USP distilled water so it is salt-free, was added to 2 x 350 ml supercompetent cells. The operation was performed in a cuvette with a diameter of 0.2 cm, according to the following protocol: 2.5 kV, 200 ohm, 25 F.

After electroporation the cells were carefully transferred into 2 x 25 ml of SOC medium, incubated for 30 minutes at 100 rpm, at 37 °C, then a sample was taken, a sequence was diluted from it and dripped onto [LB] , [LB; 100 μg/ml ampicillin] and [LB; 10 g/ml tetracycline] plates, and it was grown overnight at 37 °C. The same procedure was followed in the case of non-electroporated control products and control products electroporated with water. After taking a sample, the 2 x 25 ml culture was infected with 2 x 250 μΐ M13K07 helper phage, shaken at 37 °C for 30 minutes at 220 rpm, and then the whole product was

inoculated. The 2 x 250 ml [2YT; 100 μς/πιΐ ampicillin; 30 μg/ml kanamycin] culture was grown in two 2-litre flasks at 37 °C, at 220 rpm, for 18 hours. On the basis of titration our library contained 7 x 10 variants .

Example 2: Selection of the library on MASP-1 and MASP-2 target enzymes

2.1. The target enzymes

Human MASP-targets consist of a serine-protease (SP) domain and two complement control protein domains (CCP-1,-2) (Gal 2007). These are recombinant fragment products, which carry the catalytic activity of the entire molecule. The proteins were produced in the form of inclusion bodies, from which the conformation with biological activity was obtained by renaturation . Purification was performed by anion and cation exchange separation. The activity of the proteins was tested in a solution and also in a form linked to the ELISA plate. (For the precise details of production see Ambrus 2003) .

The data of the targets used during selection:

MASP-1 CCP1-CCP2-SP: Mw = 45478 Da, c _stock = 0.58 g/1

(hereinafter MASP-1) .

MASP-2 CCP1-CCP2-SP : Mw = 44017 Da, c _st0 c _k = 0.45 g/1

(hereinafter MASP-2) Anti-Flagtag antibody: c _st0 c _k = 4 g/1, (Sigma, Monoclonal ANTI-FLAG M2 antibody produced in mouse, cat# F3165)

2.2. Steps of selection

2.2.1. Isolating the phages

At the end of the operation described in chapter 1.3, phages were produced in 2 x 250 ml of culture for 18 hours. In the first step of the selection they were isolated to be able to use the library immediately for display.

The cell culture was centrifuged at 8,000 rpm for 10

minutes, at 4 °C. The supernatant, which contained

bacteriophages, was poured into clean centrifuge tubes, and a precipitating agent l/5 ^th of its volume was added to it [2.5 M NaCl; 20% PEG-8000] . Precipitation took place at room temperature, for 20 minutes. Then it was centrifuged again at 10,000 rpm for 15 minutes, at 4 °C. The

supernatant was discarded, it was centrifuged again for a short time, and the remaining liquid was pipetted off. The white phage precipitate was solubilised in 25 ml [PBS; 5 mg/ml BSA; 0.05% Tween-20] buffer. In order to remove possible cell fragments it was centrifuged again, the supernatant was transferred into clean tubes.

2.2.2. The first selection cycle a) Immobilisation: The target molecules were immobilised on a 96-well Nunc Maxisorp ELISA plate (cat#442404 ) . During immobilisation the concentration of MASP-1 and

-2 was 20 μg/ml, and the concentration of the anti- Flag-tag antibody was 2 μg/ml. Proteins were diluted in the immobilisation buffer [200 mM Na ₂ C0 ₃ ; pH 9.4], and 100 μΐ was put in the wells. The period of immobilisation was optimised per protein. MASP-1 was incubated while mixing at 110 rev/min. at room temperature for 60 minutes, the antibody was incubated for 30 minutes, and MASP-2 was incubated overnight at

4 °C. In the first selection cycle 12 wells per target protein were used. Every second row was left empty. As negative control only immobilising buffer was put in one row. This row was then treated the same way as the ones covered with target protein.

b) Blocking: The immobilising solution was removed, and 200 μΐ/well of blocking buffer [PBS; 5 mg/ml BSA] was put onto the plate. It was incubated at room temperature, while mixing it at 150 rev/min. for at least 1 hour.

c) Washing: The ELISA-plate was washed 4 times using 1 1 of wash buffer [PBS; 0.05% Tween-20] .

d) Selection: The phages of the library isolated as described above were pipetted onto the plate, 100 μΐ in each well. It was incubated at room temperature, while mixing it at 110 rev/min., for 2.5 hours.

e) E. coli XL1 Blue culture: During the term of the selection, XLI Blue cells were inoculated from a plate freshly picked in advance using an inoculating loop, into 2 x 30 ml of medium [2YT; 10 μg/ml tetracycline] .

These cells will be infected with phages eluted from the target proteins. At the time of infection the cells must be in the phase of exponential growth. A culture with OD ₆ oo nm ~ 0.3-0.5 was needed, which was obtained by growing it at 37 °C, at 220 rpm, for 2-3 hours .

f) Washing: The ELISA-plate was washed 12 times using 3 litres of wash buffer. g) Elution: Elution was performed using 100 mM HC1 solution, 100 μΐ/well. The acid was applied, shaken for 5 minutes, and then it was drawn from each well one by one. The phages eluted from the individual target proteins were collected in a tube, in which 12 x 15 μΐ 1 M Tris-base buffer had been put in advance to quickly neutralise the acid solution containing the phages. The tubes were immediately mixed and placed on ice .

h) Infection: 4.5 ml of XL1 Blue culture in the phase of exponential growth was put in test tubes, and it was infected with 500 μΐ of phage solution eluted from the target protein. A total number of 4 infections was performed, with phages eluted from MASP-1 and MASP-2, from the antibody and from the negative control substance. The cultures were incubated at 37 °C, at 220 rpm, for 30 minutes,

i) Titration: A 20-μ1 sample was taken from each infected culture, it was diluted to 10 times its volume with 2YT medium, and a sequence was prepared with further lOx dilutions. From each point 10 μΐ [LB; 100 μq/ l ampicillin] was dripped onto a plate and grown overnight at 37 °C.

j) Infection with helper phage: Directly after sampling, 50 μΐ M13K07 helper phage was added to each culture in the test tubes, and they were incubated for a further 30 minutes.

k) All infected cultures were transferred into 3 x 200 ml medium [2YT; 100 μg/ml ampicillin; 30 g/ml kanamycin] and incubated at 37 °C, while mixing it at 220 rpm, for 18 hours. The control substance was not treated any further, it was only needed for titration. 1) Enrichment: On the following morning titration was checked, and after only one selection cycle a large difference could be detected as compared to the control substance. The number of phages eluted from the antibody was higher by 4 orders of magnitude than the number of phages eluted from the background, in the case of MASP the difference was 1-1.5 orders of magnitude . 2.2.3. The second selection cycle

In this cycle the same steps were repeated as in the case of the first selection cycle, but in the blocking and wash buffer 2 mg/ml casein (Pierce, cat#37528) was used instead of BSA. By this modification the multiplication of phages binding to BSA can be avoided. In this step each target protein has its own control substance (12 wells), and the phages eluted and multiplied in the previous cycle were placed on each target protein.

The phages produced for 18 hours were isolated as described above, but at the end they were solubilised in 10 ml of sterile PBS buffer. The concentration of the phage solutions was measured at 268 nm, and then they were diluted with [PBS; 2 mg/ml casein; 0.05% Tween-20] buffer so that each of them has a uniform OD268 value of 0.5, and this is how they were used in the step of display. After the second selection cycle 2.7 ml of fresh exponentially growing XL1 Blue cells was infected with 300 μΐ of eluted phage. Titration was performed in all six cases (3 target proteins + 3 control substances) , and then the cultures also infected with helper phage were transferred into 30 ml [2YT; 100 g/ml ampicillin; 30 μg/ml kanamycin] medium. After the second selection cycle we obtained an enrichment of 10 ⁴ times in respect of the anti-Flag-tag antibody, 10 times in respect of MASP-1, 20 times in respect of MASP-2. 2.2.4. The third selection cycle

Everything was performed in the same way as in the case of the second cycle, casein was also kept in the buffers. After isolation the phages were solubilised in 2.8 ml of sterile PBS, and for display they were diluted to OD268 -0.5.

After the third selection cycle enormous enrichment values were obtained as compared to the control substances. The difference was 10 ⁵ times on the anti-Flag-tag antibody, and 10 ⁴ times on both MASP-s.

2.3. Testing individual clones using phage ELISA assay

In the ELISA test we were looking for phage clones that are able to bind strongly to their own target protein, while they do not display signals on the background.

a) Infection: In the case of MASP-1 and MASP-2 10 μΐ of eluted phage from selection cycle 3 was added to 90 μΐ of XL1 Blue culture in exponential phase. It was incubated for 30 minutes at 37 °C while mixing it at 220 rpm, then a 20-μ1 amount was taken out and 180 μΐ of 2YT medium was added to it. This dilution by 10 times was repeated two more times. From each dilution sequence we spread 100 μΐ on [LB; 100 μg/ml ampicillin] plates, and they were grown overnight at 37 °C. The phages eluted from the anti-Flag-tag antibody in the first selection cycle were diluted first, and only after this were the cells infected. The reason for this was that the antibody can be much more preferably immobilised on the surface of the ELISA plate, and so much more phages were eluted. Due to the high phage concentration there is the risk of one cell being infected by several phages, which results in a mixed, incomprehensible sequences.

b) Injection: into so-called "single loose" tubes, into 500 μΐ of medium [2YT; 100 g/ml ampicillin; 50 μΐ M13K07 helper phage] individual colonies were inoculated. These tubes are arranged similarly to a 96-well ELISA-plate arrangement, they move individually, so in a plate incubator, at 37 °C, while mixing at 300 rev/min they are suitable for producing small-volume cultures.

c) Immobilisation: MASP-1 and MASP-2 proteins were immobilised in a concentration of 0.01 μg/μl, while the anti-Flag-tag antibody in a concentration of 1 g/ml, in a volume of 100 μΐ/well, as described above in connection with selection, on Nunc ELISA Maxisorp plates. Each clone was tested on its own target protein, on the background and on anti-Flag-tag antibody .

d) After 18 hours the tubes were centrifuged in a rotor suitable for accommodating ELISA plates at 2,500 rpm, for 10 minutes, at 4 °C, the supernatant was pipetted into clean tubes. After ELISA the remaining supernatant, in the interest of killing off the contaminating coli bacterium was heated for 2 hours at 65 °C, After this the samples can be stored at -20 °C, and they can be used for sequencing.

e) Blocking: The liquid was removed from the immobilised samples, and 200 μΐ/well of [PBS; 2 mg/ml casein] blocking buffer was placed in each well. Incubation took place at room temperature, for at least 1 hour, while mixing at 150 rev/min.

f) Washing: The plate was washed 4 times using 1 litre of wash buffer.

g) Phage application: The phages produced and isolated as described above were diluted by 2 times using [PBS; 2 mg/ml casein; 0.05% Tween-20] buffer, and 100 μΐ was placed in the wells. From the same clone samples were pipetted into a total of 3 wells. Incubation was performed at room temperature, for 1 hour, while mixing at 110 rev/min.

h) Washing: The plate was washed 6 times using 1.5 litres of wash buffer.

i) Anti-M13 antibody: 100 μΐ of monoclonal anti-M13 HRP conjugated antibody (Amersham, cat#27-9421-01 ) diluted in [PBS; 2 mg/ml casein; 0,05% Tween-20] buffer 10,000 times was placed in the wells, and then it was incubated for 30 minutes at room temperature, while mixing it at 110 rev/min.

j) Washing: The plate was washed 6 times with 1.5 litres of wash buffer, and then twice with PBS.

k) Development: 100 μΐ of 1-Step Ultra TMB-ELISA substrate (Pierce, cat#34028) diluted to twice its amount with USP distilled water was placed in each well, shaken for a while, and then the reaction was stopped by adding 50 μΐ of 1M HCl in each well.

1) Reading: absorbance was measured at 450 nm, using BioTrak II (Amersham) plate reading photometer.

We took a sample from phage supernatants in the case of which the intensity of the background was low and which displayed at least three times more intensive signals on their own target protein, and prepared the samples for DNA sequencing. We used 2 μΐ of supernatant and used the Big Dye Terminator v3.1 cycle Sequencing Kit (Applied Biosystems ; cat#4336917) system for the PCR reaction. It was run by BIOMI Kft. (Godollo) . Example 3: results

In this example we describe the results of the tests described in examples 1-2, that is the sequences obtained. From the phages eluted from MASP-1 we tested 48 clones using ELISA, and finally we found 43 individual sequences. In the case of MASP-2 we obtained 30 individual sequences from 80 ELISA points, while in the case of the anti-Flag- tag antibody we obtained 65 interpretable individual sequences from 68 tested clones.

When interpreting the results we had to take into consideration that the NNK codon pattern used when constructing the DNA library does not ensure the same initial frequency for the individual amino acids. In the NNK codon pattern an amino acid may have one, two or three codons . The simplest way of correction is to use codon normalisation, and divide all amino acid frequencies by the number of codons the given amino acid is represented by in the NNK set. The other, more realistic approach is normalisation with the data of the sequences selected from the antibody. Not all theoretically possible sequence types can be displayed on the surface of the phages, as some of them do not result in a realisable construction, or they significantly deteriorate peptide production or the efficiency of leaving the cell. The sequence of the clones selected with anti-Flag-tag antibody reveals this non- random nature relating to producibility, so it can be used for normalisation. During normalisation, in every position the amino acid frequency values of the MASP-selected population are divided by the corresponding frequency values of the population selected on the Flag-tag antibody.

After data normalisation we made sequence logo diagrams about the sequences with the help of WebLogo accessible on the internet (http://weblogo.berkeley.edu/logo.cgi).

We examined which were the preferred amino acids in the individual positions and how much they differed from each other depending on whether they derived from MASP-1 or MASP-2.

On the basis of the results the sequences can be divided into two groups:

a) the normalised sequence diagram of the clones selected on the MASP-1 enzyme; and

b) the normalised sequence diagram of the clones selected on the MASP-2 enzyme.

The sequence logo diagrams are shown in figure 2, where the figure numbers (that is 2. a and 2.b) relate to the sequence logo diagram of the above two groups marked a) and b) , in the same order.

The sequence logo is the graphic display of the information content and amino acid distribution per position in a set of multiple aligned sequences, using the single-letter abbreviations of the amino acids. In each position the column height of the logo indicates how even the occurrence of the elements (20 different types of amino acids in our case) is. The less even this occurrence is, the higher the column. In the case of completely even distribution (all 20 amino acids occur in a proportion of 5%) the height is zero. The maximum value belongs to the case, when only one type of element (amino acid) occurs. Within the column the individual amino acids are arranged on the basis of the frequency of occurrence, the most frequent one is at the top. The height of the letter indicating the amino acid is in proportion with its relative frequency of occurrence in the given position (for example, in the case of 50% frequency of occurrence, it is half the height of the column) . In the case of colour diagrams, generally amino acids with similar chemical characteristics are shown in the same or in a similar colour, for which we used different shades of grey in the figure belonging to the present patent description.

On the horizontal axis of the sequence logo diagrams the number of the individual positions of the randomised region can be seen, site PI corresponds to position 4. On the vertical axis the information content of the positions is determined in bits.

The logo diagrams illustrate the selection taking place in the individual positions. In the initial inhibitor population the necessity to bind to the MASP enzymes resulted in intensive selection, which especially affected positions 3-6 (P2-P2' ) . It is true both in respect of the MASP-1- and the MASP-2-selected population that in the 4 positions mentioned above, from the possible 204 000 or 160 000 combinations only 8 types of sequences were selected, or at least this was the variability observed in the 4 positions among the 30-40 individual sequences. The significant reduction of the combination range is basically due to two reasons. One of these is that the spatial structure needed for efficient inhibition dictates forced conditions for the sequence. The other restriction is due to the quite narrow substrate-specificity of the MASP enzymes. The former one is illustrated by the only dominant threonine side change in position P2, which dominates this position independently from the enzyme. The reason for this is that the group is an internal structural element, which ensures the rigidity of the inhibition loop, and as a result of this it is one of the basic pillars of fulfilling the inhibitor function. At the same time, in the following positions (4-6) it can be seen how strict selection by the enzyme is asserted. The strong commitment in different directions in the same positions of the two logos was a promising sign concerning the selectivity of the inhibitors .

On the basis of the normalised sequence logos, four sequences were selected from both inhibitor populations. In the course of selection we took into consideration the outlining consensus sequence and the amino acids that occur jointly in the individual sequences. We produced these eight peptides in an isolated form in a greater amount for further tests. The names of the eight clones and the sequence of their protease binding regions are included in table 1, also stating the enzymes on which they were selected. The differences between the individual clones within the populations are marked in bold.

Table 1: a few preferred sequences according to the invention

Target Enzyme SEQ ID NO Sequence

1 MCTRKLC

2 MCTRKLCY

MASP-1

3 FCTRKLCY

4 ACTRKLCW 5 VCTRLWCE

6 VCTRLWCN

MASP-2

7 VCTRLYCN

8 VCTKL CN

For production we used an expression system created by us. For more information on it see the following chapter.

Example 4: Heterologous expression of inhibitors for quality control

4.1.1. Producing the expression vector

Due to the small size of the SGCI (35 amino acids), in itself it cannot be produced either in eukaryotic or prokaryotic systems. At the same time, in nature the desert locus efficiently separates SGTI-SGCI forms. Earlier on, at the Department of Biochemistry at Eotvos Lorand University

(ELTE) they have succeeded in producing such tandem Pacifastin forms with E. coli cells which, directed into the periplasmic space, were produced in a form of native spatial structure. In the case of this method the natural proteolytic cleavage site between the two Pacifastin units was changed to individual methionine, making use of that neither SGTI nor SGCI contains methionine. Then the two inhibitors could be processed with bromine-cyanate cleavage

(Szenthe 2004). This expression system was not acceptable for us, as it could be expected that during indicated evolution variants containing methionine would also be selected. (It did happen during MASP-1 selection.) For this reason we created a new system.

We cloned the SGCI variants in pMal-p2G vector to obtain a construction with the following arrangement: PSS - MBP - poliN linker - TEV cleavage site - SGCI variant - Stop where

PSS means periplasmic signal sequence;

MBP means Maltose Binding Protein, E. coli's own periplasmic protein, as a fusion partner;

poliN linker means an asparagin distance keeping member consisting of ten members in the manufacturer's vector, between the fusion members;

TEV cleavage site means the specific recognition and cleavage site created for the TEV protease (Tobacco Etch Virus protease) ;

Stop means TAATAA translation stop codons.

4.1.2. Removing SGCI clones from the phagemid vector - PCR

The individual SGCI clones were removed from the phagemid vector used for selection, using PCR reaction. For this we used universal 5' primer, with which we inserted an EcoRI cleavage site, which is underlined in the sequence; a TEV protease recognition site, which is marked in the sequence in bold. The protease enzyme cleaves after the TEV protease recognition site. We also inserted a GlySerGly linker, which is marked in italics. The underlined part and the part in italics is on the N-terminal of the SGCI (SEQ ID NO 12) : 5' -ACTGGAATTCGAAAACCTGTATTTTCAGGGATCCGGCGAGGrGACCrGCGAACCG- 3' The 3' primers were already clone-specific, they had to be planned separately for each variant, as the randomised part was near the C-terminal. At the common part of the 3' PCR primers we inserted a Hindlll cleavage site and two stop codons in each product.

As a template we used the frozen phage supernatant used in the phage ELISA essay, 2 μΐ for a reaction of a final volume of 25 μΐ . All reagents came from the Fermentas company. The reactions were measured together following the company's instructions, annulation took place at 55 °C for 30 seconds, 25 cycles were performed with an Eppendorf Mastercycler gradient device. The PCR product was cleaned with a VioGene PCR-M kit and eluted with 30 μΐ 0.1 x EB. For creating sticky ends the PCR product was digested with EcoRI and Hindlll enzymes (10-10 U) , in 2x Tango buffer (Fermentas) in a final volume of 50 μΐ for 3 hours at 37 °C. The PCR product ready for ligation was cleaned again with a VioGene PCR-M kit, and it was eluted with 30 μΐ 0.1 x EB.

4.1.3. Ligation and vector preparation The recipient vector (pMal-p2G) was opened under the same conditions as the PCR product, with EcoRI and Hindlll enzymes, 1 ug plasmid was used. The digested product was run in 0.8% agarose gel, then it was cut out from there and isolated with a QIAgen Gel Extraction kit and eluted with 50 ul 0.1 x EB.

For ligation the following components were prepared:

10 μΐ digested vector (about 1 μg)

10 μΐ digested PCR product 5 μΐ 10x ligase buffer (NEB)

24 μΐ distilled water

1 μΐ T4 ligase (NEB, 400ϋ/μ1)

The reaction was incubated for 2 hours at room temperature.

4.1.4. Transformation

Using the entire ligation reaction we transformed XL1 Blue cells as described in the chapter on library construction. After selecting a few of the drown colonies for the following day, plasmid was isolated as described above. The integration of the insert was tested with a PCR reaction performed with an insert and a vector specific oligo pairs. Positive constructions were also tested with sequencing.

4.1.5. Protein production Transformation

2 μΐ expression vector

100 μΐ BL21 star competent cell

The cells were incubated on ice for 30 minutes, and then for 1 minute they were exposed to a heat shock at 42 °C. 200 μΐ LB medium was added to the cells, it was shaken for 30 minutes at 37°C, and then 100 μΐ of it was spread on an LB/agar + ampicillin (camp=100 ug/ml to the end) plate. The plate was incubated overnight at 37 °C.

Preparing an initial culture

On the following day, using a sterile pipette end from a separate colony we inoculated some into 3 ml LB/ampicillin medium, and it was shaken overnight at 37 °C. Production

From the saturated initial culture we inoculated 2 ml into 1 litre LB/ampicillin medium, divided it into 3 two-litre test-tubes and shook it at 37 °C, until it reached the value OD600=0.5. It took about 4 hours. At this point we induced the cells with an IPTG solution of a final concentration of 0.3 mM, and shook it for 4 more hours at 37 °C. Then the cells were centrifuged (10 minutes, 6000 rpm, 4°C), the supernatant were poured off and the cells were suspended in 80 ml of ice-cold 1 mM MgC12 solution.

Purification

Rough fractioning

The cell suspension was divided into two 50 ml falcon tubes and frozen overnight at -20 °C. On the following day the cells were defrosted at room temperature, and immediately after this they were centrifuged (10 minutes, 10, 000 rpm, 4°C) and the supernatant was kept. This is the periplasm containing fusion protein. During freezing the cytoplasm of the cells is protected by the cell wall, but a smaller part of the cells is unavoidably extruded and genomic DNA gets into the periplasm. The DNA content was removed with general nuclease treatment and then with salting out. 20U Benzonase nuclease (Novagen) was added to the periplasm per ml and incubated overnight at room temperature. On the following day the fusion protein was salted out with 90% saturated ammonium sulphate and the precipitated proteins were centrifuged (10 minutes, 10,000 rpm, 4°C) . The precipitate was suspended in 70 ml 2.5 mM HC1 solution, and it was dialysed for two times one hour in 2 litres of 2.5 mM HC1 solution. At a low pH value the majority of the contaminating protein is precipitated, and the precipitation was removed by centrifuging (10 minutes, 15000 rpm, 4 °C) .

Processing TEV protease cleavage took place under the following conditions :

100 mM TrisHCl pH 7.6

50 μΜ DTT

20 μg mL TEV protease

The reaction was incubated at room temperature for two hours. We produced the TEV protease ourselves on the basis of the publication of van den Berg, 2006. For cleavage we did not add a reducing agent to the solution to protect the disulphide bridges of the SGCI variants. The DTT present in the solution derives from the storage buffer of the TEV protease. The cleavage was tested with 15% SDS PAGE method.

Isolation of SGCI variants In this phase, in the solution there is only MBP and inhibitor mostly separated from each other. The inhibitor was isolated using reversed-phase HPLC procedure, on a Phenomenex Jupiter C4 300A type, 250x10 mm semi-preparative column. The sample was prepared filtered through a 0.22 urn sterile filter, and then it was taken to a column equilibrated with 0.1% trifluoroacetic acid (TFA) / distilled water solution (solution A) . For separation we used acetonitrile (HPLC grade)/ 0.08% TFA solution (solution B) , the gradient was 1.3%/minute . Besides an eluent flow rate of 2 ml/minute the inhibitor variants were eluated at a retention time between 25 and 30 minutes, depending the amino acid sequence. Besides 220 ran the process could also be detected with 280 nm UV light absorption, as all clones produced by us contained Trp or Tyr side chains. Separation was realised with HP1100 type HPLC system. Agilent ChemStation software was used for system control, data collection and evaluation.

Quality control - HPLC/MS

In the case of all isolated inhibitors mass spectrometry was used for quality control. Mass spectrometry analysis was realised with HP1100 type HPLC-ESI-MS system, with flow-injection method, using 10 mM ammonium formate, pH 3.5 solution. The settings of the device were the following. Both the drying and the pulverising gas was nitrogen, the flow rate of the drying gas was 10 1/minute, its temperature was 300 Celsius degrees. The pressure of the pulverising gas was 210 kPa, the capillary voltage was 3500 V. The total ion current (TIC) chromatogram was recorded in positive ion setting within the range of 100-1500 mass/charge. The mass data were evaluated with Agilent ChemStation software.

The abbreviations of the individual inhibitors produced, the sequence and mass data of the randomised region are included in table 2.

Table 2: The theoretical and measured molecular weights of a few peptide inhibitors produced with chemical synthesis, according to the invention Theoretical Measured

SEQ ID NO Sequence

weight (Da) weight (Da)

1 MCTRKLCW 4083, 6 4083

2 MCTRKLCY 4060, 6 4060

3 FCTRKLCY 4076, 6 4076

4 ACTRKLCW 4023, 5 4024

5 VCTRL CE 4052, 5 4052

6 VCTRL CN 4037, 5 4037 ^"

7 VCTRLYCN 4014,5 4014

8 VCTKL CN 4009, 5 4008

4.2. Determining Ki constants on MASP enzymes

The inhibition constant of all eight inhibitor variants produced was measured on both enzymes. For determining the Ki constants we used catalytic enzyme fragments also used for selection (CCP1-CCP2-SP) . The synthetic substrate used in the measurements was Z-L-Lys-SBzl hydrochloride (Sigma, C3647), from which a 10 mM stock solution was prepared. The reactions were performed in a volume of 0.5 ml, at room temperature, in a buffer consisting of [20 mM HEPES; 145 mM NaCl; 5 mM CaCl ₂ ; 0.05% Triton-X100 ] . The substrate cleaved by the enzyme entered into a reaction with the dithiodipyridine auxiliary substrate (Aldrithiol-4 , Sigma, cat#143057) present in the solution in 2x excess. The release of the chromophore group created in this way was monitored in a spectrophotometer at 324 nm. A dilution sequence was prepared from the individual inhibitors, the enzyme was added to it, and it was incubated for 1 hour at room temperature. The concentration of the substrate and the length of the measuring period were chosen so that under the given conditions the enzyme should consume less than 10% of the substrate. In the course of measuring, a measuring method developed for the characterisation of tight-binding inhibitors was used (Empie, 1982) . The incline of the straight line drawn on the initial phase of the reaction was normalised with the incline received in the case of the uninhibited enzyme reaction, and multiplied with the initial or total enzyme concentration. As a result of this we obtained the free enzyme concentration, which was shown as a function of the initial inhibitor concentration and drawn according to the following equation :

E = y = Eo- (Eo+x+Ki- ( ( (E ₀ +x+Ki) ^Λ 2) -4*E ₀ *x) ^Λ (1/2) ) /2, where E is the free (uninhibited) enzyme concentration, and Eo is the initial enzyme concentration. The MASP-1 and MASP- 2 concentration was determined by titration with CI inhibitor. The results were calculated as the average of parallel measurements. The results are summarised in table 3.

Table 3: Summarising table of the enzyme inhibition of the individual inhibitors. In the sequences shown the bold letters have the same meaning as in table 2.

K _± MASP- K _± MASP-

Target SEQ ID Selectivity

Sequence 1 2

enzyme NO (Ki/Ki)

(nM) (nM)

1 MCTRKLCW 19.8 6 000 303

2 MCTRKLCY 14 36 000 2714

MASP-1

3 FCTRKLCY 6.8 58 000 8529

4 ACTRKLC 27.2 20 000 735 5 VCTRLWCE 153 000 49.1 3116

6 VCTRL CN 87 000 35 2486

MASP-2

7 VCTRLYCN 176 000 32.3 5449

8 VCTKL CN n . m 6

On the basis of the inhibition data it can be said that we have succeeded in producing inhibitors stronger than all other inhibitors produced so far against both MASP enzymes. In the case of the strongest inhibitors an increase in affinity by 10 times was reached on the MASP-1 enzyme and by 30 times on the MASP-2 enzyme as compared to the most intensively inhibiting SFTI-based variants.

A further significant result, besides the result that the appropriate SGPI-2-based inhibitors inhibit MASP-1 more intensively than SFTI-based ones, are mostly inefficient with respect to the MASP-2 enzyme, even a nearly 104 times difference was detected in the inhibition constants. It means that we have succeeded in producing completely selective MASP-1 inhibitors in a practical sense.

4.3. The effect of peptides on the three complement activation pathways

The complement system can be activated through three pathways and it leads to the same single end-point. Three activation pathways include the classical, the lectin and the alternative pathway. MASP-s are the enzymes of the initial phase of the lectin pathway, so it is important to know what effect the MASP inhibitors developed by us have on the lectin pathway, on the other two activation pathways and on the joint phase following the meeting of the three pathways .

For measuring we used the so-called IELISA kit (Euro- Diagnostica AB, COMPL300) developed for the selective measuring of the complement pathways, on the basis of the instructions for use attached to the kit. The guiding principle of measuring is that according to the three activation pathways it uses three measuring conditions, in which the currently examined complement activation pathway can operate, while the other two pathways are inactive. At the same time, the product detected during measuring is not a pathway-selective component, but the last element of the joint section of the activation pathways, the C5-9 complex.

For measuring, the blood sample was incubated for 1 hour at room temperature, then it was centrifuged and the serum was stored in small batches at -80 °C. The serum was diluted according to the prescriptions with the buffer belonging to the given complement pathway, it was incubated for 20 minutes at room temperature, the dilution sequence prepared from inhibitors was added to it, it was incubated for 20 minutes at room temperature, then it was pipetted into the appropriate wells of a special ELISA plate. In the following, washing, incubation and antibody addition was performed according to the protocol. It was incubated for 20 minutes with the substrate, and then the data was read at 450 nm in a spectrophotometer. 100% activity was represented by the serum without an inhibitor. The measurements were performed at the same time and on the same plate, from one single melted serum sample. The measurements lead to the result that the inhibitors selected on the MASP-2 enzyme are efficient and specific inhibitors of the lectin pathway of the complement system. This result is in compliance with the result demonstrated earlier, according to which these inhibitors inhibit the MASP-2 enzyme very efficiently, which enzyme, according to our present knowledge, is responsible for the initiation of the lectin pathway. The inhibitor variants selected on the MASP-1 enzyme produced an unexpected result. These variants, without any exception, also proved to be efficient and specific inhibitors of the lectin pathway. Although in recent times an increasing number of experimental results indicated the important complementary role of MASP-1 in the activation of the lectin pathway (Takahashi 2008), up until now it has remained a generally accepted view that the MASP-2 enzyme is responsible for the activation of the lectin pathway, while MASP-1 only increases it. The most obvious explanation of our observation is that MASP-1 activates MASP-2, so MASP-1 has a key role in the activation of the lectin pathway. Further experiments are needed to prove our hypothesis. The inhibitor concentrations (IC50) needed for reducing the uninhibited lectin pathway activity by half are included in table 4, where we also stated the Ki values mentioned above and their proportion.

Table 4: The inhibitor concentrations (IC50) needed for reducing the uninhibited lectin pathway activity by half, the Ki values, and the proportion of the inhibitor concentrations (IC50) needed for reducing the uninhibited lectin pathway activity by half and the Ki values.

the

Target IC50 IC50/Ki

SEQ ID NO Sequence target

enzyme (nM)

enzyme

(nM)

1 MCTRKLC 39 19.8 2

2 MCTRKLCY 28 14 2

MASP-1

3 FCTRKLCY 39 6.8 5.7

4 ACTRKLC 39 27.2 1.4

5 VCTRL CE 103 49.1 2.1

6 VCTRLWCN 110 35 3.1

MASP-2

7 VCTRLYCN 134 32.3 4.1

8 VCTKL CN 57 6 9.5

Numerous serine proteases operate in the complement system, and some of them are very similar to the ASP enzymes. Despite this our inhibitors did not inhibit either the classical or the alternative pathway.

As in the course of measuring the classical and the alternative pathway the presence of the inhibitors did not inhibit the creation of the terminal C5-9 complex, it is for certain that the peptides do not inhibit the proteases of the joint section of the complement system, so the inhibition of the lectin pathway really took place at the beginning of the lectin pathway, at the level of the MASP enzymes. It is worth pointing out that the IC50 data obtained in the course of the WIELISA measuring is about 2- 10 times higher than the Ki values obtained in the course of MASP-2 inhibition measurements based on synthetic substrates. A possible explanation for this is the following. The inhibitor peptides bind to the MASP-2 enzyme directly at the substrate binding site, and this binding successfully competes with the relatively weak interaction of small synthetic substrates with the same enzyme surface. However, besides the substrate binding site situated on the protease domain, physiological substrates can create bonds via other surfaces too (exosites), and they bind to the enzyme with a higher affinity than small synthetic substrates. It is because of this higher affinity that inhibitor molecules must be used in a higher concentration for the balance to be shifted from the enzyme-substrate complex towards the enzyme-inhibitor complex.

We find it a fact of outstanding importance that as compared to SFTI inhibitors, from SGPI-2-based inhibitors 100 times lower inhibitor concentration is enough to reduce the activity by half. It may have a great practical significance in the course of experiments performed later on living systems, where a typical task may involve setting the inhibitor concentration inside the entire vascular system of a living organism so that the given MASP enzyme is completely inhibited.

4.4. The effect of peptides on blood coagulation We performed blood coagulation measurements using blood plasma taken from healthy individuals. From the blood obtained through venipuncture and treated with sodium citrate (3.8% wt/vol) the plasma was isolated by centrifugation (2,000 g, 15 minutes, Jouan CR412 centrifuge) .

Prothrombin time (PT) testing the extrinsic pathway of blood coagulation was measured on Sysmex CA-500 (Sysmex, Japan) automatic system using Innovin Reagent (Dale Behring, Marburg, Germany) . Activated partial thromboplastin time (APTT) testing the intrinsic pathway of blood coagulation and thrombin time (TT) directly testing thrombin operation was measured on a Coag-A-Mate MAX (BioMerieux, France) analyser using TriniClot reagent (Trinity Biotech, Wichlow, Ireland) and Reanal reagent (Reanal Finechemical , Hungary) . The effect of the inhibitors on blood coagulation was measured in a final concentration of 5 uM, which is 40-90 times the IC50 values determined in the case of MASP-2 inhibitors and ~125 times the IC50 value determined in the case of MASP-1 inhibitors in the WIELISA test.

On the basis of the results it can be clearly stated about MASP-2 inhibitors that in the measuring concentration they are inefficient with respect to all proteases of the blood coagulation cascade. It complies with our knowledge according to which MASP-2 and blood coagulation proteases have no common physiological substrate. From this aspect MASP-1 is different, as it has several known common substrates with thrombin: fibrinogen, coagulation factor XIII or the PAR-4 receptor (Krarup 2008, Megyeri 2009) . Despite all this, in a concentration of 5 uM none of the MASP-1 inhibitor variants inhibited the activity of thrombin, which is clearly indicated by the unchanged nature of the thrombin times. These inhibitors have no effect on the extrinsic pathway of blood coagulation, which is proved by that the prothrombin times are also the same as the uninhibited control. There is one significant variant (SEQ ID NO 3) that does not influence the intrinsic pathway either, so this clone does not have an effect on any of the blood coagulation components. At the same time three variants (SEQ ID NO 2, SEQ ID NO 1, and SEQ ID NO 4) increased blood coagulation time detectibly on the intrinsic activation pathway. There are two possible explanations for this phenomenon. On the one part it is possible that these inhibitors, although weakly, inhibit one of factors XII, XI, IX or X. On the other part the possibility cannot be excluded that the detected effect is a consequence of the MASP-1 inhibition itself. MASP-1, as a thrombin-like enzyme, may contribute to the blood coagulation process. This effect of MASP-1 has been recently demonstrated in MASP-1 knockout transgenic mice (Takahashi 2010) . It is important to point out that this effect on the intrinsic activation pathway is only a slight effect, which can be neglected from a physiological aspect. Table 5 gives a summary of the effect of inhibitors on blood coagulation times.

Table 5: The effect of the MASP-1 and MASP2 inhibitors on blood coagulation times.

Target Thrombin APTI Prothrombin

SEQ ID NO Sequence

enzyme time (s) (s) time (s)

1 MCTRKLCW 22.3 25.97 11.1

2 MCTRKLCY 22.9 29.71 11.1

MASP-1

3 FCTRKLCY 22.5 30.03 11.0

4 ACTRKLCW 22.6 33.36 11.1

Controll 23.4 26.74 11.2

5 VCTRL CE 22.4 25.01 11.0

6 VCTRLWCN 22.8 26.31 11.1

MASP-2

7 VCTRLYCN 22.4 26.03 11.3

8 VCTKL CN 22.9 26.15 11.1

Control2 23.0 24.4 11.1 Literature references :

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SEQUENCE LISTING

<110> HUNGARIAN ACADEMY OF SCIENCES / INSTITUTE ENZYMOLOGY / EOTVOS LORAND UNIVERSITY

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<213> Artificial Sequence <220>

<223>

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Met Cys Thr Arg Lys Leu Cys

1 5

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Phe Cys Thr Arg Lys Leu Cys Tyr

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Val Cys Thr Arg Leu Trp Cys Glu 1 5

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gcggtagcga tggcaaaagc gcgtaatgct aataataata atgctaacag

50 ggtaccggtg gagg 64

<210> 11

<211> 64

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer <400> 11

gcggtagcga tggcaaaagc gcgnnktgcn nknnknnknn ktgcnnkcag

50 ggtaccggtg gagg 64

<210> 12

<211> 64

<212> DNA

<213> Artificial Sequence

<220> <223>

<400> 12

actggaattc gaaaacctgt attttcaggg atccggcgag gtgacctgcg

50

aaccg 55

<210> 13

<211> 35

<212> PRT

<213> Schistocerca Gregaria

<400> 13

Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15

Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Ala Cys Thr Leu 16 20 25 30 Lys Ala Cys Pro Gin

31 35

<210> 14

<211> 35

<212> PRT

<213> Artificial Sequence <400> 14

Glu Val Thr Cys Glu Pro Gly Thr Thr Phe Lys Asp Lys Cys Asn 1 5 10 15

Thr Cys Arg Cys Gly Ser Asp Gly Lys Ser Ala Phe Cys Thr Arg 16 20 25 30

Lys Leu Cys Tyr Gin

31 35