Patent Description:
The complement protein C5 is a central component of the complement system; a key part of the innate immune system. The complement system is an intricate immune surveillance system with numerous tasks in tightly controlled, diverse processes. It functions as a first line host defense system against infection by other organisms, and also in discriminating healthy host tissues from cellular debris and apoptotic and necrotic cells. Furthermore, it is involved in clearance of immune complexes, regulation of the adaptive immune response, promotion of tissue regeneration, angiogenesis, mobilization of stem cells and development of the central nervous system (<NPL>); <NPL>). Any trigger, for example erroneous or unrestricted activation or insufficient regulation, that disturbs the fine balance of complement activation and regulation may lead to pathologic conditions including self-attack of the host's cells leading to extensive tissue damage.

The complement system consists of about <NUM> proteins. There are three pathways to initiate complement; the classical pathway that employs Clq to recognize immune complexes on the surface of cells; the lectin pathway that is initiated when mannosebinding lectin (MBL) recognizes certain sugars; and the alternative pathway that is initiated spontaneously by hydrolysis of complement factor <NUM> (C3), a process suppressed by certain mammalian cell surface molecules not present on invading pathogens. The alternative pathway also acts as an amplification loop for the complement system. All three pathways converge at the level of C3. Cleavage of C3 into C3a and C3b leads to the formation of a convertase that in turn cleaves complement factor <NUM> (C5) into C5a and C5b. C5a is a very potent attractant of various immune cells while C5b oligomerizes with C6-<NUM> to form a pore known as the membrane attack complex (MAC) or sometimes the terminal complement complex (TCC). Activation of the complement system leads to a number of mechanisms with the purpose of neutralizing the pathogen; formation of MAC on the surface of a cell such as an invading bacteria leads to lysis, deposition of C3 and C4 cleavage products C3b and C4b aids opsonization leading to phagocytosis of the pathogen by macrophages and anaphylatoxins such as C3a and C5a attracts monocytes and neutrophils to the site of activation, up-regulates surface markers leading to increased immunologic susceptibility and to the release of cytokines.

C5 is a <NUM>-kDa glycoprotein comprised of <NUM> disulfide-linked polypeptide chains, alpha and beta, with a molecular mass of <NUM> and <NUM> kDa, respectively (<NPL>). <NPL>) constructed the complete cDNA sequence of human complement pro-C5, which is predicted to encode a <NUM>,<NUM>-amino acid pro-molecule that contains an <NUM>-amino acid leader peptide and a <NUM>-amino acid linker separating the beta and alpha chains (SEQ ID NO: <NUM>). Since C5 is common to all pathways of complement activation, blocking C5 will stop the progression of the cascade regardless of the stimuli and thereby prevent the deleterious properties of terminal complement activation while leaving the immunoprotective and immunoregulatory functions of the proximal complement cascade intact.

The complement system's key role in the defense against pathogens in general makes it an interesting target for pharmaceutical intervention. This is emphasized by the fact that many mutations or impaired regulation of complement is involved in various diseases and conditions. These include increased susceptibility to auto-immune diseases such as systemic lupus erythematosis (SLE) where deposition of immune complexes triggers the classical pathway (<NPL>). In addition, mutations of the complement proteins C1-C5 often result in SLE or SLE like symptoms. Other autoimmune diseases with a strong involvement of the complement system are rheumatoid arthritis (RA) where immune complexes may activate complement in the RA joint, Sjögren's syndrome, dermatomyositis and other autoantibody driven diseases such as Guillain-Barré syndrome (GBS), Fisher syndrome (<NPL>), different types of vasculitis, systemic sclerosis, anti-glomerular basement membrane (anti-GBM) and anti-phospholipid syndrome (APS) (<NPL>). Furthermore, complement inhibition have been proven effective in animal models of such different conditions as periodontitis (<NPL>), wound healing (<NPL>on), tumor growth (<NPL>) and diseases of the eye such as uveitis and age-related macular degeneration (AMD) (<NPL>).

Antibodies targeted to human complement C5 are known from, e.g., <CIT>; <CIT>; and <CIT>. Eculizumab (Soliris™) is a humanized monoclonal antibody directed against protein C5 and prevents cleavage of C5 into C5a and C5b. Eculizumab has been shown to be effective in treating paroxysmal nocturnal hemoglobinuria (PNH), a rare and sometimes life threatening disease of the blood characterized by intravascular hemolytic anemia, thrombophilia and bone marrow failure, and is approved for this indication. Eculizumab was also recently approved by the FDA for treatment of atypical hemolytic syndrome (aHUS), a rare but life threatening disease caused by loss of control of the alternative complement pathway leading to over-activation manifested as thrombotic microangiopathy (TMA) leading to constant risk of damage to vital organs such as kidney, heart and the brain. In aHUS, transplantation of the damaged organ only temporarily helps the patient as the liver continues to produce the mutated form of controlling protein (most often complement factor H or other proteins of the alternative pathway). A related disease with a transient acute pathophysiology is HUS caused by infection of Shiga toxin positive E. coli (STEC-HUS) and there are promising clinical data suggesting efficacy also for this condition (<NPL>). Finally, the C5 blocking antibody Eculizumab has proven efficacious in preventing antibody mediated rejection (AMR) in recipients of highly mismatched kidneys (<NPL>), and in treating autoimmune neuropathies such as neuromyelitis optica and myasthenia gravis (<NPL>; <NPL>).

Apart from full length antibodies, single-chain variable fragments (scFV), minibodies and aptamers targeting C5 are described in literature. These C5 inhibitors may bind to different sites (epitopes) on the C5 molecule and may have different modes of action. For example, whereas Eculizumab interacts with C5 at some distance of the convertase cleavage site, the minibody Mubodina® interacts with the cleavage site of C5. The C5 inhibitory protein Ornithodoros moubata Complement Inhibitor (<NPL>) from soft tick Ornithodoros moubata has been hypothesized to bind to the distal end of the CUB-C5d-MG8 superdomain, which is close to the convertase cleavage site (<NPL>). In contrast to the three proteins mentioned above inhibiting cleavage of C5, the monoclonal antibody TNX-<NUM> binds to a C5a epitope present both on intact C5 and released C5a without inhibiting the cleavage of C5.

C5 binding polypeptides, comprising a C5 binding motif, are disclosed in the International Patent Application No. <CIT>, published as <CIT>. In particular, <CIT> discloses a C5 binding motif, BM, consisting of the amino acid sequence
EX<NUM>X<NUM>X<NUM>A X<NUM>X<NUM>EID X<NUM>LPNL X<NUM>X<NUM>X<NUM>QW X<NUM>AFIX<NUM> X<NUM>LX<NUM>D,.

Examples of specific C5 binding motifs, as previously disclosed in <CIT>, are shown as SEQ ID NO: <NUM>-<NUM> in the present patent application.

It is known from <CIT> that additional peptides or polypeptides may improve stabilization of C5 binding polypeptides. One example of such a polypeptide is the albumin binding domain (ABD) shown as SEQ ID NO: <NUM> in the present description. Other examples of suitable albumin binding domains are disclosed in <CIT> and <CIT>. An ABD-extended polypeptide binds to serum albumin in vivo, and benefits from its longer half-life, which increases the net half-life of the polypeptide itself (see e.g. <CIT>).

The continued provision of agents with comparable C5 blocking activity remains a matter of substantial interest within the field. In particular, there is a continued need for molecules that prevent the terminal complement cascade as well as the formation of the pro-inflammatory molecule C5a. Of great interest is also a provision of uses of such molecules in the treatment of disease.

It has surprisingly been found that C5 binding polypeptides and compounds, wherein the amino acid sequences have been modified in specific positions, have improved stability when compared to previously known C5 binding polypeptides and compounds.

Consequently, this invention provides a polynucleotide that encodes a polypeptide capable of binding human complement component <NUM> (C5), said polypeptide being selected from:.

The inventors have surprisingly found that modification or substitution of amino acid residue(s) in certain position(s) of the amino acid sequence of the C5 binding polypeptides as described in <CIT> improves stability of the C5 binding polypeptides while biological activity, such as binding affinity for human complement component <NUM> (C5) and inhibition of complement pathway function, is essentially retained. Thus, the biological activity of the modified C5 binding polypeptides is comparable to the biological activity of the known C5 binding polypeptides. Stability testing of the C5 binding polypeptides of the present invention demonstrates that substitution in either X<NUM>, from N to E or S, or X<NUM>, from D to E or S, improves stability. It has moreover been found that specific amino acid substitution in [L2] independently may promote stability.

The terms "C5 binding" and "binding affinity for C5" as used in this specification refers to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance technology, such as in a Biacore instrument (GE Healthcare). C5 binding affinity may e.g. be tested in an experiment in which C5 is immobilized on a sensor chip of a Biacore instrument, and the sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing C5, or fragment thereof, is passed over the chip. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding of the polypeptide to C5. If a quantitative measure is desired, for example to determine the apparent equilibrium dissociation constant KD for the interaction, surface plasmon resonance methods may also be used. Binding values may for example be defined in a Biacore <NUM> instrument (GE Healthcare). C5 is immobilized on a sensor chip of the measurement, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected over the chip. KD values may then be calculated from the results using for example the <NUM>:<NUM> Langmuir binding model of the BIAevaluation software provided by the instrument manufacturer. The C5 or fragment thereof used in the KD determination may for example comprise the amino acid sequence represented by SEQ ID NO: <NUM>. Examples of how C5 binding affinity may be tested are given herein, see Example <NUM> and <NUM>.

In a preferred form of the invention, said polynucleotide encodes a polypeptide selected from:.

As previously disclosed in <CIT>, the C5 binding polypeptide encoded by a polynucleotide according to the invention may form part of a three-helix bundle protein domain. Said C5 binding motif [BM] essentially may form part of two alpha helices, with an interconnecting loop, within said three-helix bundle. A second interconnecting loop, referred to herein as [L2], connects the C5 binding motif to the third alpha helix, referred to as the "Backbone".

In one embodiment, the C5 binding motif [BM] encoded by the polynucleotide is essentially as disclosed in <CIT>. However, according to the present invention the C5 binding motif encoded by the polynucleotide preferably consists of <NUM>, rather than <NUM>, amino acids, and may in addition carry further amino acid substitutions.

In a further preferred aspect, [BM] in the polypeptide encoded by the polynucleotide comprises or consists of an amino acid sequence selected from the group consisting of positions <NUM>-<NUM> in SEQ ID NOS: <NUM>-<NUM>. More preferably, [BM] comprises or consists of the amino acid sequence shown as positions <NUM>-<NUM> in SEQ ID NO: <NUM>.

In preferred forms of the invention, at least one of the following eighteen, optionally nineteen, conditions is fulfilled by the encoded polypeptide:.

More preferably, at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen or nineteen of the above conditions are fulfilled.

In an embodiment, X<NUM> and X<NUM> are independently selected from E and S. Preferably, (a) X<NUM> is S and X<NUM> is E, or (b) X<NUM> is E and X<NUM> is S.

In an embodiment, X<NUM> is S and X<NUM> is D.

In another embodiment, X<NUM> is N and X<NUM> is E.

In a further aspect, the polypeptide encoded by the polynucleotide according to the invention comprises the amino sequence shown as SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, or SEQ ID NO: <NUM>.

In a further aspect, there is provided a polynucloetide that encodes a compound capable of binding C5, said compound comprising:.

Preferably, said albumin binding domain comprises the amino acid sequence shown as SEQ ID NO: <NUM>.

Preferably, when X<NUM> is D, a preferred compound comprises or consists of the amino sequence shown as SEQ ID NO: <NUM>. When X<NUM> is E, preferred compounds comprise or consist of the amino sequence shown as SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM> or SEQ ID NO: <NUM>. When X<NUM> is A, a preferred compound comprises or consists of the amino acid sequence shown as SEQ ID NO: <NUM>. In the above listed amino acid sequences of the C5 binding compounds, amino acid residues <NUM>-<NUM> represent the amino acid sequence of a C5 binding polypeptide, residues <NUM>-<NUM> represent the amino acid sequence of a linker, and residues <NUM>-<NUM> represent the amino acid sequence of an albumin binding domain.

In an embodiment, the linking moiety is absent.

As discussed above, preferred C5 binding polypeptides encoded by the polynucleotides according to the invention include those wherein X<NUM> and X<NUM> are independently selected from E and S. Specifically, compounds according to the invention can be derived from PSI0242 (SEQ ID NO: <NUM>) but have modifications in at least one of positions <NUM>, <NUM> and <NUM>. For instance, as shown in <FIG> and the sequence listing, the preferred compound designated PSI0378 (SEQ ID NO: <NUM>) carries the amino acid substitutions N52S, D53E and D60E; the preferred compound designated PSI0379 (SEQ ID NO: <NUM>) carries the amino acid substitutions N52S, D53E and D60A; the preferred compound designated PSI0381 (SEQ ID NO: <NUM>) carries the amino acid substitutions N52E, D53S and D60E; and the preferred compound designated PSI0383 (SEQ ID NO: <NUM>) carries the amino acid substitutions N52S, D53E and D60E. Further, SEQ ID NO: <NUM> also carries substitutions in the loop [L2], namely D36R, D37Q and S39E. Moreover, the preferred compound designated PSI0403 (SEQ ID NO: <NUM>) carries the amino acid substitutions D53E and D60E, and the preferred compound designated PSI0404 (SEQ ID NO: <NUM>) carries the amino acid substitutions N52S and D60E.

As accounted for above, the inventors have surprisingly found that amino acid substitutions in certain positions of the amino acid sequence of the C5 binding polypeptides as described in WO <NUM>/<NUM> may improve stability. Such substitutions improve stability of the C5 binding compounds while biological activity, such as C5 binding capability and inhibition of hemolysis in vitro, is retained. Stability testing of the C5 binding compounds of the present invention demonstrate that for instance each of N52S (X<NUM>) and D53E (X<NUM>) (SEQ ID NO: <NUM>) individually, as well as removal of D60 (X<NUM>) (SEQ ID NO: <NUM> lacking linking moiety) improves stability. The combination of the substitutions N52S, D53E and D60E or D60A further improves the stability (SEQ ID NO: <NUM> and SEQ ID NO: <NUM>). Each of the combined substitutions of N52S and D60E (SEQ ID NO: <NUM>) and D53E and D60E (SEQ ID NO: <NUM>) has similarly been found to improve stability. This indicates that each of the listed amino acid substitutions is involved in improving the stability of the polypeptide, and thus that each of these substitutions will provide further stabilized C5 binding polypeptides and compounds compared to previously known C5 binding polypeptides and compounds.

However, the skilled person will be able to identify polypeptides and/or compounds which have modifications in at least one of positions <NUM>, <NUM> and <NUM>, and/or in the loop [L2], but which also carry additional modifications like substitutions, small deletions, insertions or inversions, and nevertheless have substantially the disclosed biological activities and improved stability. Further, a C5 binding polypeptide and/or compound encoded by the polynucleotides according to the invention could comprise further C terminal and/or N terminal amino acids that improve production, purification, stabilization in vivo or in vitro, coupling, or detection of the polypeptide.

It has been found that removal of D60 or an amino acid substitution in position <NUM> of SEQ ID NO: <NUM> alone improves stability of the C5 binding compounds encoded by the polynucleotides of the invention compared to previously known C5 binding compounds. Preferably, the linking moiety is KVEGS (X<NUM>=E) while X<NUM>X<NUM> may be ND, and an example of a preferred compound carrying such a linking moiety is PSI0410 (SEQ ID NO: <NUM>). In another preferred embodiment, D60 and the entire linking moiety is absent and an example of such a compound is the preferred compound designated PSI0369 (SEQ ID NO: <NUM>).

In embodiments of the above aspect, [BM] and the albumin binding domain are as defined above in related aspects. Preferably, [L2] is DDPS.

Specific amino acid substitutions in the loop [L2] have been found to improve stability of the C5 binding compounds (e.g. SEQ ID NO: <NUM>) encoded by the polynucleotides of the invention compared to previously known C5 binding compounds. In embodiments of the above aspect, said [BM] and albumin binding domain are individually as defined above in related aspects. Preferably, said linking moiety is a peptide comprising the amino acid sequence KVX<NUM>GS, wherein X<NUM> is selected from D, E and A.

The invention further includes vectors, such as expression vectors, comprising polynucleotides according to the invention. Included are also host cells which comprise such vectors.

The polynucleotides according to the invention encode C5 binding polypeptides and compounds for use in therapy. In particular, the C5 binding polypeptides and compounds are useful in methods for the treatment and/or prophylaxis of C5-related conditions, such as inflammatory diseases; autoimmune diseases; infectious diseases; cardiovascular diseases; neurodegenerative disorders; graft injury; eye diseases; kidney diseases; pulmonary diseases; haematological diseases such as paroxysmal nocturnal hemoglobinuria (PNH); allergic diseases and dermatological diseases.

In methods for treatment and/or prophylaxis, the said C5 binding polypeptide or compound can preferably be administered intravenously, subcutaneously, by inhalation, nasally, orally, intravitreally, or topically.

The C5 binding compound designated PSI0242 (SEQ ID NO: <NUM>) was formulated in <NUM> NaP /<NUM> NaCl pH <NUM> and subjected to an accelerated stability study for <NUM> weeks at <NUM>. The stability was measured by the appearance of new variants after the stability testing by SDS-PAGE and Reversed Phase HPLC (RPC). In both analyses the initial sample and the one subjected to the stability study were run in parallel. For the SDS-PAGE, <NUM>µg protein was loaded into each well. The RPC was run on an Agilent <NUM> HPLC using a Mobile Phase A consisting of <NUM> % trifluoroacetic acid (TFA) in water, and using a Mobile Phase B consisting of <NUM> % TFA / <NUM> % MeOH / <NUM> % isopropylamine (IPA) / <NUM> % water.

The results show that new forms of the protein are formed during incubation, these new forms visualized as bands in SDS-PAGE (<FIG>) and as new peaks in Reversed Phase HPLC (RPC) chromatograms (<FIG>). In <FIG>, the main peak after <NUM> weeks incubation corresponds to <NUM> % of the original protein sample.

Positions <NUM>-<NUM> in SEQ ID NO: <NUM> correspond to the polypeptide Z06175a, previously disclosed in <CIT>as SEQ ID NO: <NUM>. PSI0242 (SEQ ID NO: <NUM>) was produced essentially as disclosed in <CIT>.

Modified C5 binding polypeptides and compounds were synthesized and purified essentially as described in <CIT>.

Briefly, DNA encoding C5 binding Z variants was E. coli codon optimized and synthesized by GeneArt, GmbH. The synthetic genes representing the C5 binding Z variants were subcloned and expressed in E.

Intracellularly expressed Z variants were purified using conventional chromatography methods. Homogenization and clarification was performed by sonication followed by centrifugation and filtration. Anion exchange chromatography was used as capture step. Further purification was obtained by hydrophobic interaction chromatography. The purifications were executed at acidic conditions (pH <NUM>). Polishing and buffer exchange was performed by size exclusion chromatography.

The purified proteins were formulated in <NUM> NaP /<NUM> NaCl pH <NUM> and subjected to an accelerated stability study for <NUM> weeks at <NUM>. The stability was measured by the appearance of new variants after the stability testing by SDS-PAGE and Reversed Phase HPLC (RPC). In both analyses the initial sample and the one subjected to the stability study were run in parallel. For the SDS-PAGE, <NUM>µg protein was loaded into each well. An example of a resulting gel is shown in <FIG>.

The RPC was run on an Agilent <NUM> HPLC using a Mobile Phase A consisting of <NUM> % trifluoroacetic acid (TFA) in water, and a Mobile Phase B consisting of <NUM> % TFA / <NUM> % MeOH / <NUM> % isopropylamine (IPA) / <NUM> % water. An example of a resulting chromatogram is shown in <FIG> for SEQ ID NO: <NUM>.

The results of the stability testing are summarized in Table I, below.

It can be concluded from Table I that certain modified C5 binding polypeptides or compounds have improved properties, such as increased stability, when compared with PSI0242. Such improved C5 binding polypeptides or compounds include PSI0334 (SEQ ID NO: <NUM>), PSI0340 (SEQ ID NO: <NUM>), PSI0369 (SEQ ID NO: <NUM>), PSI0377 (SEQ ID NO: <NUM>), PSI0378 (SEQ ID NO: <NUM>), PSI0379 (SEQ ID NO: <NUM>), PSI0381 (SEQ ID NO: <NUM>), PSI0383 (SEQ ID NO: <NUM>), PSI0400 (SEQ ID NO: <NUM>), PSI0410 (SEQ ID NO: <NUM>), PSI0403 (SEQ ID NO: <NUM>) and PSI0404 (SEQ ID NO: <NUM>). In six of the mentioned variants (SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>), the amino acid residues in positions <NUM>-<NUM> have been substituted from ND (cf PSI0242) to SE. In SEQ ID NO: <NUM>, the corresponding substitution is from ND to ES. In SEQ ID NO: <NUM> only the amino acid residue in position <NUM> has been substituted from D to E, while in SEQ ID NO: <NUM> the amino acid residue in position <NUM> has been substituted from N to S.

Further, PSI0378 (SEQ ID NO: <NUM>), PSI0381 (SEQ ID NO: <NUM>), PSI0383 (SEQ ID NO: <NUM>), PSI0410 (SEQ ID NO: <NUM>), PSI0403 (SEQ ID NO: <NUM>) and PSI0404 (SEQ ID NO: <NUM>) have in common an amino acid residue substitution from D to E in position <NUM>.

The combined benefit of stability enhancing substitutions in position <NUM> or <NUM> and position <NUM> can be seen in <FIG>, showing the chromatogram of PSI0383 (SEQ ID NO: <NUM>). In PSI0379 (SEQ ID NO: <NUM>) the substitution in position <NUM> is from D to A.

In PSI0369 (SEQ ID NO: <NUM>) the linker moeity (including D60) is altogether removed, yielding a more stable C5 binding compound and indicating the influence of position <NUM> upon stability of the C5 binding compounds.

Human serum albumin was immobilized to Amine Reactive <NUM>nd generation (AR2G) Dip and Read Biosensors (Pall Life sciences (ForteBio) Cat # <NUM>-<NUM>) by amine coupling. PSI0242 (SEQ ID NO: <NUM>; <NUM>) and C5 binding compounds (<NUM>) in read buffer (HBS-EP Buffer ready-to-use <NUM>, GE Healthcare #BR100188) were loaded, each onto a separate sensor with HSA, for <NUM> seconds followed by a base line recording for <NUM> seconds in read buffer before being subjected to human C5 (Quidel Cat # <NUM>) in concentrations ranging from <NUM> to <NUM> in read buffer with a regeneration cycle and a base line recording between each concentration. Regeneration conditions for the sensors were <NUM> Glycine, pH <NUM> (three pulses with <NUM> seconds and running buffer for <NUM> seconds). Each spectrogram was reference subtracted against an analogous construct containing an albumin binding domain (SEQ ID NO: <NUM>) but without the C5 binding capacity. The data were analyzed according to Langmuir <NUM>:<NUM> model using ForteBio Analysis <NUM> (Pall Life sciences (ForteBio) kinetics software).

The KD of the interaction with C5 relative to PSI0242 (SEQ ID NO: <NUM>) is shown in Table II. The KD of PS10242 varied from <NUM>-<NUM> in different runs.

The results in Table II indicate that C5 binding compounds according to the invention have a binding capacity to human C5 which is similar to that of the polypeptide PSI0242 (SEQ ID NO: <NUM>) disclosed in <CIT>.

A chemically synthesized PSI0400 (SEQ ID NO: <NUM>) was ordered from BACHEM AG. The stability of the polypeptide was tested according to the same methodology as in Example <NUM>. The results of the stability testing are shown in Table III.

The stability of the PSI0400 was comparable to the polypeptides that were produced in E. coli in Example <NUM>.

The integrity of the fold of PS10400 (SEQ ID NO: <NUM>) was compared to a recombinant C5 binding polypeptide (PSI0257, SEQ ID NO: <NUM>), produced in accordance with the methods of Example <NUM>, using far UV circular dichroism (CD) spectra.

The CD spectra were recorded by a J-<NUM> CD spectropolarimeter (Jasco, Japan). The samples were diluted to <NUM>/ml protein concentration using Pi buffer (<NUM> Na-K-PO<NUM>, pH <NUM>). A CD spectrum of Pi buffer was firstly recorded, then spectra were recorded for each of the samples and lastly for the Pi buffer again. As the two buffer spectra coincide, the firstly recorded spectrum was used as the buffer spectrum. The buffer spectrum was smoothened using the Savitzky-Golay procedure with convolution width of <NUM>. The other spectra were smoothened according to the same procedure with a convolution width of <NUM>. The smoothened buffer spectrum was then subtracted from each of the other smoothened spectra. The CDNN program was used to estimate the secondary content of the proteins and the resulting estimations are presented in Table IV. The results showed that neither the two amino acid substitutions at position <NUM> and <NUM> nor the polypeptide production by chemical synthesis influence the secondary structure content of the chemically synthesized polypeptide. The integrity of the secondary structure content was compared to the recombinantly produced PST0257 (SEQ ID NO: <NUM>).

The binding affinity of the C5 binding compounds PSI0242 (SEQ ID NO: <NUM>), PSI0340 (SEQ ID NO: <NUM>), PSI0378 (SEQ ID NO: <NUM>), and PSI0410 (SEQ ID NO: <NUM>) and the C5 binding polypeptide PSI0400 (SEQ ID NO: <NUM>) for human C5 was analyzed using a Biacore T200 instrument (GE Healthcare). Human C5 (A403, Quidel Corporation) was coupled to a CM5 sensor chip (<NUM> RU) using amine coupling chemistry according to the manufacturer's protocol. The coupling was performed by injecting hC5 at a concentration of <NUM>µg/mL in <NUM> Na-acetate buffer pH=<NUM> (GE Healthcare). The reference cell was treated with the same reagents but without injecting human C5. Binding of the C5 binders to immobilized hC5 was studied with the single cycle kinetics method, in which five concentrations of sample, typically <NUM>, <NUM>, <NUM>, <NUM> and <NUM> in HBS-EP buffer (<NUM> HEPES pH <NUM>, <NUM> NaCl, <NUM> EDTA, <NUM>% Surfactant P20, GE Healthcare) were injected one after the other at a flow rate of <NUM>µL/min at <NUM> in the same cycle without regeneration between injections. Data from the reference cell were subtracted to compensate for bulk refractive index changes. In most cases, an injection of HBS-EP was also included as control so that the sensorgrams were double blanked. The surfaces were regenerated in HBS-EP buffer. Kinetic constants were calculated from the sensorgrams using the Langmuir <NUM>:<NUM> analyte model of the Biacore T200 Evaluation Software version <NUM>. The resulting KD values of the interactions are tabulated in the Table V.

The stability enhancing amino acid substitutions are not detrimental for the ability of the molecules to bind to C5 and thus do not influence their biological activities.

For studies of classical complement pathway function and inhibition thereof by the C5 binding compounds PSI0378 (SEQ ID NO: <NUM>) and PSI0410 (SEQ ID NO: <NUM>), and C5 binding polypeptide PSI0400 (SEQ ID NO: <NUM>), sheep erythrocytes were prepared from fresh sheep whole blood in Alsever's solution (Swedish National Veterinary Institute) and thereafter treated with rabbit anti-sheep erythrocyte antiserum (Sigma) to become antibody sensitized sheep erythrocyte (EA). The whole process was conducted under aseptic conditions. All other reagents were from commercial sources.

The in vitro assay was run in <NUM>-well U-form microtiter plate by consecutive additions of a test protein, a complement serum and EA suspension. The final concentrations of all reagents, in a total reaction volume of <NUM>µL per well and at pH <NUM>-<NUM>, were: <NUM> CaCl <NUM>; <NUM> MgCl <NUM>; <NUM> NaN <NUM>; <NUM> NaCl; <NUM> % gelatin; <NUM> sodium barbital; <NUM> barbituric acid; <NUM> million EA; complement protein C5 serum at suitable dilution, and C5 binding compound or polypeptide at desired concentrations.

The C5 binding compounds and polypeptide were pre-incubated with the above described complement serum for <NUM> on ice prior to starting the reaction by the addition of EA suspension. The hemolytic reaction was allowed to proceed at <NUM> during agitation for <NUM> and was then optionally ended by addition of <NUM>µL icecold saline containing <NUM> % Tween <NUM>. The cells were centrifuged to the bottom and the upper portion, corresponding to <NUM>µL supernatant, was transferred to a transparent microplate having half-area and flat-bottom wells. The reaction results were analyzed as optical density using a microtiter plate reader at a wavelength of <NUM>.

On all test occasions, a control sample (PSI0242, SEQ ID NO: <NUM>) and vehicle were included in each plate to define values for uninhibited and fully inhibited reactions, respectively. These values were used to calculate the % inhibition of the complement hemolysis at any given sample concentration. The inhibitory potencies (IC <NUM>-values) of tested C5 binding compounds and polypeptide were defined by applying the same assay in the presence of a controlled concentration of human C5 added to C5 depleted serum. For highly potent inhibitors (low nanomolar to sub-nanomolar), a final C5 concentration of the reaction mixture was controlled at <NUM>, which was optionally established by using C5 depleted or deficient sera. The results are presented below in Table VI.

The results from the hemolysis assay show that the improved C5 binding compounds SEQ ID NO: <NUM> and <NUM> are comparable to the reference compound. The C5 binding polypeptide SEQ ID NO: <NUM> was functional in the assay but since it does not contain an albumin binding domain the results cannot be directly compared to the reference compound.

For assessment of C5 binding compounds binding affinity for albumin, a human albumin ELISA utilizing recombinant human albumin (coating) and commercially available antibodies (primary and detecting) purchased from Novozymes, Affibody AB and DakoCytomation, respectively, was used. A method standard prepared from PSI0242 (SEQ ID NO:<NUM>), comprising a C5 binding polypeptide and an albumin binding domain of streptococcal protein G, was used for quantification of samples.

A <NUM>-well microplate was coated with recombinant human albumin. The plate was then washed with phosphate buffered saline containing <NUM> % Tween <NUM> (PBST) and blocked for <NUM>-<NUM> hours with <NUM> % casein in PBS. After a plate wash, the standard, method controls, control sample and test samples are added to the plate. After incubation for <NUM> hours, unbound material was removed by a wash. A goat Anti-Affibody® IgG (Affibody AB, cat no. <NUM>) was added to the wells and the plate was incubated for <NUM> hours to allow binding to the bound C5 binding compounds. After a wash, rabbit anti-goat IgG HRP was allowed to bind to the goat antibodies for <NUM>. After a final wash, the amount of bound HRP was detected by addition of TMB substrate, which was converted to a blue product by the enzyme. Addition of <NUM> hydrochloric acid after <NUM> minutes stopped the reaction and the color of the well contents changed from blue to yellow. The absorbance at <NUM> was measured photometrically, using the absorbance at <NUM> as a reference wavelength. The color intensity was proportional to the amount of PSI0242 (SEQ ID NO:<NUM>) and the sample concentrations were determined from the standard curve.

The C5 binding compounds comprising an albumin binding domain of streptococcal protein G proved capable of binding to human albumin and the data is presented in Table VII below.

The results from the assay showed that both of the investigated stability improved C5 binding compounds maintain their ability to bind human albumin.

The C5 binding polypeptides/compounds that showed an improved stability compared toPS10242 in the <NUM> weeks stability test at <NUM> (Example <NUM>) were subjected to a longer <NUM> month stability test at <NUM>. The setup of the stability test was as described in Example <NUM> and the evaluation of the stability was made by measuring the main peak of the chromatogram percentage of the total protein content by Reversed Phase HPLC (RPC), the RPC method was performed as described in example <NUM>. The <NUM> weeks data from example <NUM> is included in Table VIII below to make the interpretation easier.

C5 binding compounds with amino acid substitutions in position <NUM>, <NUM> from ND to SE and a replacement in position <NUM> from D to E or A (SEQ ID NO: <NUM>, <NUM>, and <NUM>) compared to PSI0242 have a higher proportion of protein in the original form after <NUM> months at <NUM> than PSI0242 (SEQ ID NO: <NUM>) has after <NUM> weeks at the same conditions. The other compounds also displayed an increased stability.

Previously known polypeptide variants derived from protein Z (<NPL>) with binding affinity for other target molecules than C5 were similarly modified in specific positions of the amino acid sequence in order improve stability. Selection and production of the original polypeptide variants with binding affinity for the human epidermal growth factor receptor <NUM> (HER2), the plateletderived growth factor receptor beta (PDGF-Rβ), the neonatal Fc receptor (FcRn), and the carbonic anhydrase IX (CAIX) is disclosed in e.g. <CIT>, <CIT>, <CIT>, and <CIT>. The stability improved polypeptide variants were produced by site-directed mutagenesis at selected positions of the amino acid sequence. The stability improving amino acid substitutions in the polypeptide variants Z02891 (SEQ ID NO: <NUM>), targeting HER2; Z15805 (SEQ ID NO: <NUM>), targeting PDGF-Rβ; Z10103 (SEQ ID NO: <NUM>), targeting FcRn; and Z09782 (SEQ ID NO: <NUM>), targeting CAIX, are specified below in Table IX. These stability improved polypeptide variants differ from the C5 binding polypeptides of the present invention for example in that they have a binding motif [BM] with binding affinity for HER2, PDGF-Rβ, FcRn, and CAIX.

All variants were cloned with an N-terminal <NUM> x Histidine-tag (His6) and the achieved constructs encoded polypeptides in the format MGSSHHHHHHLQ-[Z#####]. Mutations were introduced in the plasmids of the polypeptide variants using overlapping oligonucleotide primer pairs encoding the desired amino acid substitutions and by applying established molecular biology techniques. The correct plasmid sequences were verified by DNA sequencing.

E coli (strain T7E2) cells (GeneBridge) were transformed with plasmids containing the gene fragments encoding the original and the modified polypeptides. The cells were cultivated at <NUM> in TSB-YE medium supplemented with <NUM>µg/ml kanamycin and protein expression was subsequently induced by addition of IPTG. Pelleted cells were disrupted using a FastPrep®-<NUM> homogenizer (Nordic Biolabs) and cell debris was removed by centrifugation. Each supernatant containing the polypeptide variant as a His6-tagged protein was purified by immobilized metal ion affinity chromatography (IMAC) using His GraviTrapTM columns (GE Healthcare) according to the manufacturers instructions. Purified polypeptide variants were buffer exchanged to phosphate-buffered saline (PBS; <NUM> KH2PO4, <NUM> Na2HPO4, <NUM> NaCl, <NUM> KCl, pH <NUM>) using PD-<NUM> desalting columns (GE Healthcare). The correct identity of each polypeptide was verified by SDS-PAGE and HPLC-MS.

Apart from the substitutions of one of (SEQ ID NO: <NUM>-<NUM>) or both of (SEQ ID NO: <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>) N52 and D53, substitutions were also performed in the positions corresponding to the loop [L2]. Thus, in the polypeptide variants of SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, [L2] is RQPE.

For carrying out the stability testing, the polypeptide variants, formulated in PBS pH <NUM>, were diluted to <NUM>/ml and <NUM>µl aliquotes were incubated at <NUM> for <NUM> weeks. Samples collected prior to and after the stability test were analyzed by SDS-PAGE using <NUM>% Bis-Tris NuPAGE gels (Invitrogen) and loading <NUM>µg protein into each well. Resulting Coomassie blue stained gels are shown in <FIG>. The stabilty was assessed by the appearance of new variants after incubation at the elevated temperature and mutated variants were compared to respective original polypeptide.

All polypeptide variants with the modifications as outlined in Table IX showed improved stability compared to the respective original polypeptide in the sense that a second band just above the main band observed for samples of the original polypeptide was not visible in samples of the modified polypeptides with the substitution D53E and/or N52S, see <FIG>. Polypeptides with the substitutions D53E and/or N52S combined with the substitutions D36R, D37Q and S39E showed similar profiles on the SDS-PAGE gel. The substition D53E alone or in combination with the substitutions D36R, D37Q and S39E seemed to reduce the amount of the specie with an alternative confirmation observed as a second band on the SDS-PAGE gel, but could not completely prevent the formation of this species.

In addition, the binding capability of the modified polypeptide variants was tested. All polypeptide variants retained their binding affinity for their target after being modified (results not shown).

Claim 1:
A polynucleotide that encodes a polypeptide capable of binding human complement component <NUM> (C5), said polypeptide comprising the amino acid sequence:
[BM]-[L2]-QSX<NUM>X<NUM>LLX<NUM>EAKKLX<NUM>X<NUM>X<NUM>Q
wherein, independently of each other,
[BM] is a C5 binding motif comprising the amino acid sequence:
EX<NUM>X<NUM>X<NUM>A X<NUM>X<NUM>EIDX<NUM>LPNLX<NUM>X<NUM>X<NUM>QWX<NUM>AFIX<NUM>X<NUM>LX<NUM>;
wherein, independently of each other,
X<NUM> is selected from H, Q, S, T and V;
X<NUM> is selected from I, L, M and V;
X<NUM> is selected from A, D, E, H, K, L, N, Q, R, S, T and Y;
X<NUM> is selected from N and W;
X<NUM> is selected from A, D, E, H, N, Q, R, S and T;
X<NUM> is selected from A, E, G, H, K, L, Q, R, S, T and Y;
X<NUM> is selected from N and T;
X<NUM> is selected from I, L and V;
X<NUM> is selected from A, D, E, H, K, N, Q, R, S and T;
X<NUM> is selected from I, L and V;
X<NUM> is selected from D, E, G, H, N, S and T;
X<NUM> is selected from K and S;
X<NUM> is selected from A, D, E, H, N, Q, S, T and Y
[L2] is selected from DDPS and RQPE;
X<NUM> is selected from A and S;
X<NUM> is selected from N and E;
X<NUM> is selected from A, S and C;
X<NUM> is selected from E, N and S;
X<NUM> is selected from D, E and S, provided that X<NUM> is not D when X<NUM> is N; and
X<NUM> is selected from A and S.