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WO2012095519A1 - Inhibiteurs puissants de l'activation du complément - Google Patents

Inhibiteurs puissants de l'activation du complément Download PDF

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Publication number
WO2012095519A1
WO2012095519A1 PCT/EP2012/050494 EP2012050494W WO2012095519A1 WO 2012095519 A1 WO2012095519 A1 WO 2012095519A1 EP 2012050494 W EP2012050494 W EP 2012050494W WO 2012095519 A1 WO2012095519 A1 WO 2012095519A1
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WIPO (PCT)
Prior art keywords
complement
factor
inhibitor
cells
comp
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Ceased
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PCT/EP2012/050494
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English (en)
Inventor
Stefan Heinen
John D. Lambris
Nadine LAUER
Christine Skerka
Peter F. Zipfel
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Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie
Leibniz Institut fuer Naturstoff Forschung und Infektionsbiol eVi
Original Assignee
Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie
Leibniz Institut fuer Naturstoff Forschung und Infektionsbiol eVi
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Publication of WO2012095519A1 publication Critical patent/WO2012095519A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21043—Classical-complement-pathway C3/C5 convertase (3.4.21.43)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21047—Alternative-complement-pathway C3/C5 convertase (3.4.21.47), i.e. properdin factor B
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention provides a chimeric recombinant polypeptide that directs inhibitors to the side of damage and complement activation.
  • Said recombinant polypeptide is a chimeric recombinant polypeptide comprising the C-terminal surface bind- ing region of complement factor H (CFH) of at least one or even two up to six short consensus repeats (SCR) or analogs thereof derived from the CFHR molecules linked with an inhibitor of the complement allowing inhibition of complement activation.
  • CCH complement factor H
  • SCR short consensus repeats
  • the present invention relates to methods allowing prophylaxis or treatment of complement-mediated diseases, disorders or conditions, like inflammatory diseases or autoimmune diseases, during transplantation or other kind of surgery to protect blood vessels as well as combating overshooting complement activation in a subject, comprising the step of administering the potent inhibitor according to the present invention.
  • the complement system is a central part of innate immunity and plays an im- portant role in the elimination of microbes, clearance of damaged self cells, cellular homeostasis and also in modulating the adaptive immune response.
  • Activation of the complement system occurs via three major pathways, the alternative pathway (AP), the classical pathway (CP) and the lectin pathway (LP).
  • AP alternative pathway
  • CP classical pathway
  • LP lectin pathway
  • Each pathway is initiated by unique activators and generates a C3 convertase that cleaves the central complement component C3 into C3a and C3b.
  • C3b is spontaneously and continuously activated by "tickover" of C3 and in the absence of inhibitors activated C3 is deposited on any surface. This molecule forms additional convertases and amplifies the reaction.
  • TCC terminal complement complex
  • the AP is spontaneously and constantly activated while the CP and LP are activated by immune complexes or by carbohydrates expressed e.g . on bacteria.
  • an amplification loop is formed which utilizes the C3 con- vertase of the AP.
  • the initial steps of AP activation are non-discriminatory and occur on any surface.
  • the discrimination between activator- and non-activator surfaces is mediated by potent and highly specific regulators and inhibitors.
  • the activated AP has disastrous effects and initiates central effector functions that from side of the host need to be targeted onto foreign surfaces (e.g. microbes or damaged self cells) but at the same time need to be restricted on host surfaces. Restriction of AP activation on host cell and tissue surfaces is mediated by regulators and inhibitors. These regulators are distributed in fluid phase, such as plasma and body fluids or are expressed as integral membrane proteins to form non-activator surfaces.
  • Factor H the key regulator of the AP, is a 150 kDa abundant plasma protein that regulates the AP in fluid phase and that also attaches to cellular surfaces.
  • Factor H is composed of 20 consecutive protein domains, termed short consensus repeats (SCRs).
  • the protein has two central functional regions located at the opposite ends of the protein.
  • the four N-terminal domains SCR1 -4 form the regulatory region that regulates the C3 conver- tase of the alternative pathway.
  • SCR1 -4 accelerate the decay of a preformed C3bBb conver- tase or act as a cofactor for Factor I mediated C3b cleavage and inactivation.
  • the C- terminus represented by SCRs 15-20 is the central surface attachment or recognition region. This part of the protein contacts cellular surfaces, binds to heparin, to surface glyco- saminoglycans and also to C3b and to C3d.
  • Factor H uses the C-terminal attachment region to bind e.g. to the surface of renal endothelial cells, retinal pigment epithelial cells, the glomerular basement membrane of the kidney or the Bruch's membrane of the retina.
  • This type of surface recognition and attachment is highly specific and directs the complement inhibitor to self surfaces and in particular to self surfaces where complement activation is initiated.
  • Factor H controls complement activation at those surfaces.
  • the relevance of this surface attachment is highlighted by diseases associated gene mutations that results e.g. in the renal disease atypical Hemolytic Uremic Syndrome (aHUS). The majority of HUS associated gene mutations cluster in the C-terminal surface attachment region and consequently reduce Factor H surface binding.
  • aHUS atypical Hemolytic Uremic Syndrome
  • Compstatin is an excellent and effective complement inhibitor that allows therapeutic intervention of complement mediated diseases (Rick- lin, D. and J. D. Lambris, 2008, Advances in experimental medicine and biology 632:273-292.)
  • Compstatin is a cyclic peptide 1 3-residues in size, initially identified by screening a phage-display library, for peptides that inhibit complement by binding to native C3 and thereby blocking subsequent conversion, as well as the amplification of all three pathways.
  • Compstatin shows low toxicity, no adverse effects in humans as demonstrated by in-vitro and in-vivo studies, and binds specifically to human and primate C3.
  • Compstatin prolongs the lifetime of a porcine-to-human xenograft and prevents complement activation on biomaterials e.g .
  • Compstatin and analogs thereof are described in US 6,31 9,897, WO2004/026328 A1 , WO2007/062249, WO2007/044668 or WO2008/97525.
  • the inhibitory activity of Compstatin was improved more than 260 times during a 1 0 year optimization process of the original peptide using chemical, biophysical and computational approaches (Katragadda, M., et.al., J Med Chem, 2006; 49(1 5) : p. 461 6-22).
  • Potentia Pharmaceuticals successfully completed a phase I clinical studies for the treatment of AMD.
  • One of such targeted inhibitor molecules links the human complement receptor CR2 with the N-terminal domains of Factor H (CR2-fH, Taligen Therapeutics) or CR2 to mouse Crry (CR2-Crry) (Atkinson, C. et al. , 2005. The Journal of clinical investigation 1 1 5:2444-2453; Huang, Y. et al., 2008, J Immunol 1 81 :8068-8076.).
  • the two modular proteins use the CR2 region to target the inhibitor to the site of complement activation and C3b formation and use the regulatory region of Factor H for local complement inhibition.
  • the chimeric proteins according to the present invention demonstrate improved binding and inhibitory effects regarding C3 binding as well as by comparing the regulatory/inhibitory activities in fluid phase and on two different cellular surfaces, such as erythrocytes and nucleated cells.
  • the chimeric proteins according to the present invention allow to inhibit complement activation both on activator and non- activator surfaces. Bound to the surface of living as well as on nucleated cells, like necrotic nucleated cells, the chimeric polypeptides according to the present invention protected surfaces from complement mediated damage and the tagged complement inhibitor was more than 30 % more efficient than the untagged complement inhibitor.
  • tagged means the presence of the surface binding region of factor H, namely at least one, two, three, four, five or even up to six of the C-terminal short consensus repeats (SCR), preferably three, four, five or six of said SCRs, of Factor H or analogs thereof, in particular, the SCRs of the analogs CFHR1 (SCR 3, 4, 5), CFHR2 (SCR 3, 4), CFHR3 (SCR 3, 4), CFHR4 (SCR 3, 4 or only 3) and CFHR5 (SCR 3, 4) as well as of SCR 6 and 7 of CFHL1 .
  • SCR C-terminal short consensus repeats
  • the polypeptides according to the present invention containing the complement inhibitor tagged with the C-terminal short consensus repeats derived from factor H or analogs thereof increases the affinity of the inhibitor almost ten fold. Consequently, the polypeptides according to the present invention allow a more potent complement control at the sites of immune stress, i.e. complement activation by targeting the inhibitor specifically to surfaces at the desired sites, thus, abolishing at least some of the adverse effects occurring upon systemic complement inhibition.
  • polypeptides according to the present invention are particularly useful for local treatment or prophylaxis of complement mediated diseases, disorder, or conditions allowing local complement inhibition.
  • the recombinant chimeric polypeptide according to the present invention consists of an inhibitor of the complement and at least one or even two up to six of the SCR of Factor H.
  • Said inhibitor is in particular an inhibitor of the C3 conver- tase, C5 convertase or an inhibitor of the terminal complement complex.
  • the inhibitor of the complement is preferably selected from Compstatin, Efb, Sbi and SCIN.
  • the inhibitor of the complement comprises at least the complement inhibitory domain of the respective complement inhibitor.
  • isolated nucleic acid molecules encoding the polypeptides according to the present invention, vectors containing said nucleic acid molecules as well as host cells containing the vector or the nucleic acids of the present invention are described.
  • pharmaceutical compositions comprising the polypeptides, nucleic acid molecules, vectors and/or cells according to the present invention, optionally, containing additionally a pharma- ceutically acceptable diluent, excipients or carrier, are disclosed. Said polypeptides are particularly useful for the treatment of diseases or disorders comprising an overshooting complement activation or complement-mediated diseases, disorders or conditions.
  • the method according to the present invention comprises the administration of pharmaceutical compositions comprising the recombinant chimeric polypeptide, the nucleic acid, factors or host cells, accordingly.
  • Said methods allow the prophylaxis or treatment of complement-mediated diseases, disorders or conditions, like inflammatory diseases or autoimmune diseases, during transplantation or other kind of surgery to protect blood vessels as well as combating overshooting complement activation in a subject, comprising the step of administering the potent inhibitor according to the pre- sent invention.
  • the present invention relates to coatings for devices and coated devices containing the recombinant polypeptide according to the present invention.
  • C3, C3b or heparin were coated onto a microtiter plate (each at 5 ⁇ g / ml), saturated with blocking buffer and then increasing amounts of either COMP CFH15-20, SCR15-20 or the control protein BSA were added. Bound proteins were detected by using a HRP labeled polyclonal goat Factor H antiserum.
  • COMP CFH15-20 black squares
  • COMP CFH15-20 bound dose- dependently to C3. At all concentrations > 2.5 ⁇ g/ml COMP CFH15-20 showed significantly stronger binding to C3 as compared to the untagged deletion mutant SCR15-20 (open squares).
  • COMP_CFH 15-20 binds C3b significantly stronger as compared to SCR15-20 for all tested concentrations.
  • Efb_SCR18-20, Efb or SCR18-20 was assayed by ELISA using a monoclonal anti-histidin specific antibody.
  • Efb_SCR18-20 bound to C3b and C3d.
  • Untagged Efb and SCR18-20 bound to C3b and C3d to a lesser degree than the chimera.
  • Efb_SCR18-20 binding to C3d was more pronounced than to C3b.
  • COMP_CFH 15-20 showed an increased binding efficiency than SCR15-20 and 4W9A and an improved dissociated than 4W9A.
  • erythrocytes which represent activator surfaces, were incubated in 7.5 % complement active NHS together with increasing amounts of COMP CFH 15-20 or SCR15- 20 for 30 min at 37 °C. The amount of lysed rE was determined by measuring the absorb- ance of the supernatant at 414 nm.
  • COMP CFH15-20 black squares
  • COMP CFH15-20 prevented lysis of rE in a dose-dependent manner.
  • This protective effect was significantly stronger as compared to SCR15-20 (open squares), which did not affect lysis of rE.
  • the Compstatin analog 4W9A (black column) showed a comparable protection of lysis as COMP CFH15-20 whereas the linearized inactive Compstatin (hatched column) variant showed no protective effect on the lysis of rE.
  • the control peptide 4W9A completely prevented C5a generation.
  • the linearized negative control peptide did not protect from complement activation therefore a high C5a amount is detectable.
  • the figures A-B show one representative experiment of three independent experiments with three replicates each. Error bars indicate SD. * , P ⁇ 0.05; ** , P ⁇ 0.01 ; *** , P ⁇ 0.001 .
  • the inhibitory activity of COMP CFH15-20 was also assayed for sheep erythrocytes (sE) that represent non-activator surfaces.
  • sE sheep erythrocytes
  • HSACFH inhibitor Factor H
  • Incubation of sE with COMP SCR15-20 (black squares) in the presence of HSACFH for 30 min at 37 °C protected the erythrocytes from lysis. The effect was dose dependent.
  • Figure 5 COMP CFH15-20 enhances metabolic activity of living nucleated CHO cells upon complement challenge
  • NHS complement active human serum
  • the viability value for CHO cells in the presence of NHS after 5h incubation was set to 100 %.
  • the data represent mean values and standard deviation for three independent experiments. Error bars indicate SD. * , P ⁇ 0.05; ** , P ⁇ 0.01 ; P ⁇ 0.001 .
  • COMP_CFH 15-20 and SCR15-20 bind to the surface of necrotic CHO cells. Binding was assayed following incubation of the cells with the appropriate proteins and bound proteins were detected with a polyclonal antiserum specific for the C-terminus of Factor H by flow cytometry. One representative experiment of three independent experiments with three replicates each is shown.
  • CHO cells were challenged with either 10 % NHS or 10 %HSACFH for 15 min at 30 °C.
  • HSACFH black histogram
  • the amount of cell bound C5b is significantly enhanced compared to cells incubated with NHS (gray histogram).
  • a representative histogram profiles from four independent experiments are shown.
  • COMP_CFH15-20 interacts also with human cell lines (e.g. HUVEC cells).
  • the binding of the complement inhibitor was assayed using flow cytometry.
  • COMP_CFH 15-20 black column
  • SCR15-20 white column
  • Unspecific antibody binding in the absence of pro- teins is shown by the dotted line.
  • One representative experiment of three independent experiments with three replicates each is shown.
  • FIG. 8 Efb_SCR18-20 displayed complement inhibitory activity in fluid phase.
  • Efb_SCR1 8-20 effect on cell vitality was analysed as follows: Efb_SCR1 8-20, SCR1 8-20 or Factor H were bound to C3b coated CHO cells and after extensive washing the cells were challenged with 1 0% HSACFH. Then cell vitality - was moni- tored Efb_SCR1 8-20 (squares) increased cellular by uptake of the non flueoresent dye resarufin and intracellular conversion to the fluorochrom resazurin. Efb_SCR1 8- 20 (squares) increased cellular enzymatic activity as marker for cell viability as revealed by the increase in intracellular conversion. Cellular viability was high when the cells were challenged with heat inactivated NHS.
  • the present invention relates to a chimeric recombinant poly- peptide, which is a chimeric recombinant polypeptide comprising the C-terminal region of complement factor H (CFH) or analogs there of comprising at least one, two, three, four, five or six, particularly preferred three, four, five or six of the short consensus repeats SCR 1 5 to 20 of CFH or the analogs of any CFHR molecule or of CFHL1 or analogs thereof, and an inhibitor of the complement allowing inhibition of complement activation.
  • CCFH complement factor H
  • SCR short consensus repeats
  • C-terminal fragment of complement factor H or analogs thereof derived from CFHR1 , CFHR2, CFHR3, CFHR4 and CFHR5 or CFHL1 herein it is meant that the polypeptide comprises at least one or even two, three, four five or six of the SCRs 1 5 to 20 of factor H, or the corresponding C-terminal domains of CFHR1 (SCR 3, 4, 5), CFHR2 ( SCR 3, 4), CFHR3 (SCR 3, 4), CFHR4 (SCR 3 or SCR 3, 4, respectively) and CFHR5 (SCR 3, 4) as well as of CFHL1 (SCR 6, 7).
  • Seq. ID No. 1 the complete sequence of factor H is provided.
  • Short consensus repeat 1 5 (Seq. I D No. 2 corresponds to aa 865 to 927 of Seq. I D No. 1
  • SCR1 6 corresponds to aa 928 to 985 (Seq. ID No. 3) of Seq. I D No. 1
  • SCR1 7 corresponds to to aa 986 to 1 043 (Seq. ID No. 4) of Seq. I D No. 1
  • SCR1 8 corresponds to to aa 1 044 to 1 1 03 (Seq. ID No. 5) of Seq. I D No. 1
  • SCR1 9 corresponds to to aa 1 1 04 to 1 1 64 (Seq. I D No. 6) of Seq. ID No. 1
  • SCR20 corresponds to to aa 1 1 65 to 1 231 (Seq. I D No. 7) of Seq. ID No. 1 .
  • the recombinant polypeptide consists of the SCR1 5 to
  • SCR1 8 to SCR20 domains.
  • SCR of Factor H any other combination of the SCR of Factor H and an inhibitor of the complement is possible.
  • the SCR may not be in the order as they naturally occur or a SCR of Factor H may be combined with a SCR of another compound.
  • polypeptide according to the present invention may comprise additionally a
  • Typical Tags are for example a His- Tag, c-myc tag etc.
  • the recombinant chimeric polypeptide according to the present invention represents a potent inhibitor of the complement, in particular an inhibitor of the C3 convertase, C5 convertase and/or an inhibitor of the terminal complement complex.
  • the term "inhibitor of the complement” comprises at least the complement inhibitory domain of an inhibitor of the complement. That is, the "inhibitor of the complement” may be a fragment of an inhibitor of the complement.
  • the bacterial inhibitors described herein may be used as fragments comprising at least the complement inhibitory domain thereof.
  • chimeric polypeptide identifies that the polypeptide is composed of at least two different components derived from different sources, i.e. different proteins.
  • the chimeric recombinant polypeptide according to the present invention is composed of the complement inhibitor Comp- statin, Efb, Sbi, and SCIN and at least three of the SCR1 5 to SCR20 short consensus repeats of factor H or of the analogs thereof derived from any one of the CFHR proteins.
  • Particularly preferred are the polypeptides of Seq. ID No. 8 to 1 1 .
  • the C-terminal fragment is composed of SCR 1 8 to 20, or of SCR 1 5, 1 7, 1 8 to 20 etc.
  • the C-terminal fragment is SCR 1 5 and 1 7 only.
  • the present invention relates to an isolated nucleic acid molecule encoding the polypeptide according to the present invention.
  • polypeptide means, according to the present invention, a peptide(s) or a (poly)peptide(s) which encompass amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds, as long as said polypeptide include the sequence noted in connection with that polypeptide.
  • peptidomi- metics of such proteins wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention.
  • a protein may comprise different protein species.
  • a protein species is (furthermore and not limiting) defined by a chemical composition and modifications of said peptide(s)/(poly)peptide(s) by, inter alia, glycosylations, acetylation, phosphorylations, lipidations or by amino acid exchanges.
  • polypeptide refers to a polypeptide of amino acids and does not refer to a specific length of the product; hence, peptides, oligopeptides and proteins are included within the definition of polypeptides.
  • polypeptides or nucleic acids may be provided in form of compositions.
  • compositions are particularly adapted for local administration to a subject.
  • said composition is in form of administration by injection or by DNA-vaccination.
  • the present invention encompasses nucleic acid molecules encoding the poly- peptides according to the present invention.
  • Said nucleic acids may be in form of single or double stranded DNA or RNA molecules.
  • molecules having altered structures, in particular being alternatively linked, e.g. by peptide bonds, are encompassed into the term nucleic acid molecules.
  • Said nucleic acids may be contained in a suitable vector.
  • Said vector may allow expression of the nucleic acids and for the polypeptide encoded by said nucleic acids.
  • Said vector maybe adapted for direct administration to a subject or may be a vector for expression of the polypeptide or the nucleic acid in known expression systems either prokaryotic or eukaryotic expression systems.
  • the expression vector is a replic- able or a non-replicable expression vector as known in the art.
  • the vectors of the present invention may be transformed or transferred into a host cell wherein the polypeptide is expressed.
  • Said host cells may be prokaryotic or eukaryotic host cells known in the art and transformation of said cells maybe effect by known methods.
  • nucleic acid molecules either encoding a polypeptide according to the present invention or an isolated antisense RNA or DNA, silencer mRNA, RNAi, mi- croRNA, non-coding RNA maybe obtained by chemical synthesis or by a known amplification technique.
  • the term "complementarity" or “complementary” between nucleic acids is the degree to which the bases in one nucleic acid strand can hydrogen bond or base pair, with the bases in a second nucleic acid strand.
  • Sufficient comple- mentary means that a sufficient number of the nucleotides in a first nucleic acid strand from base pairs with nucleotides in a second nucleic acid to generate a stable hybridization complex at about room temperature, i.e. at about 20 °C to about 25 °C.
  • Complementarity can sometimes be conveniently described by the percentage, i.e. proportion, of the nucleotides which can form base pairs between two nucleic acid strands or within a specific region or domain of the two strands.
  • the degree of complementarity can range from at least about 50 % to full, namely, 1 00 % complementarity.
  • the term "homology" as used here, is a degree of sequence identity between two nucleic acid strands or two polypeptide sequences.
  • the degree of homology can also be described by the percentage of identical nucleotides or amino acids in two nucleotides or polypeptide sequences, respectively.
  • the degree of homology between a target nucleic acid and a probe or oligonucleotide of the present invention can vary as long as selective hybridization is attained and range from at least about 50 % to about 1 00 % homology, like 60 %, 70 %, 80 %, 90 % or 95 % homology.
  • the pharmaceutical composition may be administered with a physiological acceptable carrier to a patient.
  • the term “pharmaceutically acceptable” means approved by regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particular, in humans.
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered.
  • pharmaceutically or therapeutically acceptable carrier is a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
  • a variety of pharmaceutically acceptable carriers well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer, solutions, emulsifiers, isotonic saline and pyrogen free water.
  • compositions of the present invention suitable for oral or parenteral administration may be presented as solid units such as capsules, such as tablets each containing a predetermined amount of each of the purine analog as a powder or granules or as a solution or a suspension in aqueous liquid, a non-aqueous liquid, an oil water emulsion or a water in oil liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the sub-cytostatic dosages of each of the purine analogs as described above with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniform and intimately admixing the cytostatic agent with liquid carriers or finely dividend solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • the term "therapeutically effective amount” is a dose that produces the effects for which it is administered.
  • the exact dose will depend on the focus of the treatment, and will be ascertainable by one skilled in the art using known techniques. As it is known in the art and described above, adjustment of systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the variety of the condition may be necessary and will be ascertainable with routine experimentation by those skilled in the art.
  • compositions according to the present invention can be pro- vided in suitable dosage forms.
  • dosage forms include tablets, pills, capsules, powders, granulates, salves, plasters releasing substances transdermal to the individual, tinctures, uvula, suspensions, solutions, injections, dispersions, syrups, troches, aerosols and the like.
  • dosage forms may also include injecting or implanting slow releasing devices designed specifically for the purpose or other forms of implants modified to act additionally in this fashion.
  • Slow- or controlled-release of the active ingredients may be effected by coating the same, for example with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and po- lyglycolic acids and cellulose derivative, such as hydroxypropylmethylcellulose.
  • the controlled release may be effected by using a polymer matrices, liposomes and/microspheres.
  • the pharmaceutical composition can be applied to an individual one or more times daily, weekly, monthly, or longer time intervals.
  • the composition according to the present invention can be used alone or in combination with other pharmaceutical composition.
  • the present invention relates to a method for treating a sub- ject afflicted with a disease or disorder comprising an overshooting complement activation comprising the step of administering an effective amount of a pharmaceutical composition according to the present invention, i.e. an effective amount of a recombinant polypeptide, nucleic acid molecule, vector or cell as defined herein.
  • the complement-mediated disease, disorder or condition according to the pre- sent invention may be an inflammatory disease or an autoimmune disease, in particular, said disease, disorder or conditions which can be treated with the polypeptide according to the present invention include complement mediated renal diseases, Hemolytic Uremic syndrome, MPGN I I, Asthma, Systemic Lupus Erythematosus, Antiphos- pholipid Syndrome, DEAP-HUS, Ischemia Reperfusion Injury, Macular Degeneration, Traumatic Brain Injury, as well as inflammatory conditions, that require local complement control.
  • complement mediated renal diseases Hemolytic Uremic syndrome, MPGN I I, Asthma, Systemic Lupus Erythematosus, Antiphos- pholipid Syndrome, DEAP-HUS, Ischemia Reperfusion Injury, Macular Degeneration, Traumatic Brain Injury, as well as inflammatory conditions, that require local complement control.
  • composition according to the present invention the recombinant chimeric polypeptide, the nucleic acid, the vector and/or cells according to the present invention are useful in the treatment of complement-mediated disorders, diseases or conditions, like in the prophylaxis or treatment of inflammatory diseases or autoimmune diseases.
  • the method comprises the step of administering the effective amount of the pharmaceutical composition by a route selected from the group consisting of : intravenously or by local intravitreal injection.
  • a route selected from the group consisting of : intravenously or by local intravitreal injection for example, the pharma- ceutical composition or the polypeptides is adapted to allow local administration.
  • the pharmaceutical composition including the polypeptides, nu- cleic acids, vectors or cells are a composition selected from a gel, cream, lotion or ointment suitable for application to the skin, a dry powder suitable for delivering to the lung , a sustained release formulation or device that releases the active principal over the time, a biodegradable polymeric matrix releasing the polypeptide over the time, in form of an inhalable dry powder or inhalable liquid aerosol, as a component of a nasal spray in addition DNA vaccination is an option.
  • the present invention relates to a method for inactivating or inhibiting complement activation during transplantation or dialysis comprising the step of administering a polypeptide according to the present invention or a nucleic acid according to the present invention to a subject undergoing transplantation or dialysis.
  • the present invention relates to a method for inactivating or inhibiting complement activation in body fluids comprising the step of coating surfaces or devices, like implants, which may come into contact with said body fluids with a polypep- tide according to the present invention or a nucleic acid according to the present invention.
  • the present invention relates to a coating for a device, like an implant or the like, which come into contact with body fluids, in particular, with plasma or blood, wherein said coating comprises a polypeptide according to the present in- vention or a nucleic acid according to the present invention.
  • the present invention relates to the coated device, in particular, an implant, which may come into contact with body fluids, in particular, plasma or blood, wherein said coating comprises a polypeptide according to the present invention or a nucleic acid according to the present invention.
  • the present invention relates to a method of treating an eye disorder comprising administering an effective amount of a polypeptide according to the pre- sent invention or a nucleic acid molecule according to the present invention to a subject of all suffering from the eye disorder.
  • the present invention relates to the use of the C-terminal fragments of complement factor H, CFHL1 , CFHR1 , CFHR2, CFHR3, CFHR4, CFHR5 as a targeting structure for targeting complement inhibitors to the side of complement activation.
  • HSACFH Factor H depleted serum
  • HSAC7 C7 depleted serum
  • the plasmid pBSV-SCR1 5-20 was used as template (Kuhn, S. , C. Skerka, and P. F.
  • the reaction was performed using the Phusion High Fidelity PCR Kit (NEB, Frankfurt, Germany) with the following conditions: denaturation at 98 °C for 3 min, followed by 30 cycles of denaturation at 98 °C for 1 min, annealing at 68 °C for 1 min and exten- sion at 72 °C for 1 .5 min, and a final extension of 1 0 min at 72 ° C.
  • the resulting 1 143 bp DNA fragment contained the coding region for the complete peptide of Compstatin fused to the Factor H SCR1 5-20 coding region, and is flanked by PstI and Xbal restriction sites. This DNA fragment was sub-cloned into E.
  • COMP CFH 1 5-20 was recombinantly expressed as a His-tagged protein in P. pastoris strain X33. Protein expression was induced with 1 % methanol. After 3 days of expression, the culture supernatant was harvested and the recombinant COMP CFH1 5-20 was purified by nickel affinity chromatography. The recombinant Factor H deletion construct SCR1 5-20 was expressed in the bacu- lovirus system.
  • the staphyolcoccal inhibitors Efb, Sbi and SCIN were amplified by PCR using the appropriate primers and cloned in frame into an expression vector that has the C- terminal three SCRs of Factor H inserted, (i.e. SCRs 1 8 -20). SDS-Page and Western blotting
  • Proteins were separated by SDS-PAGE and either visualized by silver staining or they were transferred onto a nitrocellulose membrane and identified with an appropriate antiserum in combination with the corresponding HRP-coupled secondary goat antiserum. Visualization occurred with the Chemiluminescence Gel Documentation System MF-ChemBIS 3.2 (Biostep, Jahnsdorf, Germany) .
  • COMP_CFH1 5-20 Binding of COMP_CFH1 5-20 to living and necrotic CHO or HUVEC cells was examined by flow cytometry.
  • Cells were washed twice in DPBS supplemented with 1 % BSA, then 20 ⁇ g / ml of COMP_CFH 1 5-20, Factor H or SCR1 5-20 was added. After extensive washing bound proteins were detected with polyclonal Factor H antise- rum and an Alexa488 labeled goat Factor H antiserum. 1 0.000 cells were routinely counted in a BD LSRI I flow cytometer and analyzed with the FACSDiva- (BD Biosciences, Heidelberg , Germany) and FlowJo software (Tree Star, Ashland, OR, USA). Living intact cells were identified as propidium iodide negative cells and necrotic cells were identified as propidium iodide positive cells (BD Biosciences).
  • the complement regulatory activity of COMP CFH 1 5-20 was analyzed in hemolytic assays with HEPES buffer (20 mM HEPES, 144 mM NaCI, 7 mM MgCI 2 , 1 0 mM EGTA, pH 7.5) using rabbit, as well as sheep erythrocytes (Rockland). Rabbit erythrocytes (rE) represent activator surfaces for NHS and are lysed due to TCC formation. 1 x1 0 7 rE were incubated with 7.5 % NHS and the effect of increasing amounts of COM P_CFH 1 5-20 was assayed after 30 min at 37 °C.
  • the fraction of lysed cells was determinated by measuring the supernatant at 414 nm and also by assaying the generation of the complement activation product C5a by ELISA (OS- TEOMedical GmbH, Bunde, Germany).
  • deposition of C5 on the surface of rE incubated in HSAC7 was detected with polyclonal C5 antiserum and examined by confocal microscopy using a laser scanning microscope LSM 51 0 META (Zeiss, Jena, Germany).
  • Identical assays were performed with sheep erythrocytes (sE) that represent non-activator surfaces and remain intact when incubated in NHS. However, these cells are lysed, when incubated in complement active HSACFH.
  • the protective effect of COMP CFH 1 5-20 on the metabolic capacity of cultivated CHO cells in the presence of HSACFH was measured using the CellTiter-Blue® assay (Promega, Mannheim, Germany).
  • the light absorbance of the CellTiter-Blue® reagent is changed by cellular reduction of the non-fluorescent dye resazurin into the fluorescent dye resorufin. Therefore, 5 x 1 0 4 CHO cells were seeded into 96-well mi- croplates (Nunc) and incubated for 2 days to allow adherence.
  • the culture medium was replaced, cells were washed with HEPES buffer and then incubated for 1 h with 20 ⁇ g / ml COMP_CFH 1 5-20, SCR1 5-20, the active and inactive Compstatin peptides lacking the Factor H tag, to allow surface binding . After an additional washing procedure the cells were treated with 5 % NHS or HSACFH. Then, the CellTiter-Blue® was added to the cells and incubated for 5 hours. During this time conversion of resazurin into resorufin was followed by measuring the absorbance of the probes at 570 nm in a microplate reader (Tecan, Crailsheim, Germany) every hour.
  • the absorption maxi- mum for resazurin is 605 nm and the absorption maximum for resorufin is 573 nm.
  • the absorbance measurements at 570 nm and using 600 nm as a reference wavelength were used to monitor the results. Values are compared to blank well containing CellTiter-Blue® reagent without cells. During all incubation steps cells were kept at 37 °C in a humidified atmosphere and 5 % C0 2.
  • the cells were treated with 1 0 % NHS or HSACFH for additional 1 5 min at 30 °C.
  • COMP_CFH1 5-20 was identified as a band with a mobility of 50 kDa.
  • the SCR1 5-20 fragment was identified as a 45 kDa band and Factor H as a 1 50 kDa band.
  • SCR1 5-20 and Factor H were identified by immunoblotting using polyclonal Factor H antiserum together with a secondary HRP-labeled antiserum.
  • COMP_CFH15-20 binds to C3 and C3b
  • C3 and C3b were immobilized and the recombinant proteins COMP_CFH 15-20 and SCR15-20 were added.
  • Both COMP_CFH15-20 and the control protein SCR15-20 used at concentrations from 2.5 - 20 ⁇ g / ml bound dose dependently to C3 ( Figure 1A).
  • COMP_CFH 15-20 well as SCR15-20 bound dose dependently to C3b ( Figure 1 B).
  • COMP CFH15-20 and the 4W9A form showed rapid complex formation and a steady state was reached in less than 1 min.
  • the tagged COMP CFH15-20 showed an enhanced relative signal intensity (C3b complex formation) and also a slower dissociation compared to 4W9A.
  • SCR15-20 showed relative weak C3b binding and thus confirms the data obtained by ELISA studies (see above).
  • a kinetic evaluation of COMP_CFH 15-20 showed a dose-depended and stable C3b binding.
  • the tagged Compstatin has two C3b binding regions and thus the interaction did not follow a 1 :1 model, but was fitted with a two- side model.
  • This approach defined two C3b binding sites of different affinity.
  • the low affinity site of 130nM corresponds to that obtained for the untagged Compstatin 4W9A, whereas the high affinity site seems to be a corresponding effect of 4W9A and SCR15-20. Therefore the chimera shows protein of Compstatin and SCR15-20 an improved C3b affinity of at least 10 times.
  • SCR15- 20 binds C3b with a K D of 5 ⁇ (data not shown).
  • the complement inhibitory activity of COMP CFH1 5-20 was determined by using rE (rabbit erythrocytes) that represent activator surfaces for NHS and that are therefore lysed upon incubation with NHS.
  • COMP CFH1 5-20 inhibited lysis of rabbit erythrocyte and this effect was dose-dependent ( Figure 3A).
  • SCR1 5-20 did not affect lysis of rE.
  • the untagged Compstatin (4W9A) also inhibited lysis but the linear, inactive, Compstatin did not affect rE lysis.
  • both the tagged COMP_CFH 1 5-20 and the untagged Compstatin (4W9A) used at a concentration of 20 ⁇ g / ml showed comparable effects.
  • the complement inhibitory effect of COMP CFH15-20 was also analyzed by following generation of the complement activation product C5a in the supernatant.
  • COMP_CFH15-20 and the untagged Compstatin (4W9A) showed comparable effects on C5a generation at concentration of 20 pg/ml.
  • COMP_CFH 15-20 also affected C5b deposition on the surface of rabbit erythrocytes, as assayed by immunofluorescence microscopy.
  • C7 depleted human serum HSAC7
  • COM P_CFH 15-20 inhibited C5b deposition on the rE surface as indicated by the weak fluorescence signal.
  • SCR15-20 complement is activated and C5b is deposited onto the surface, as indicated by a strong fluorescence signal.
  • the untagged Compstatin (4W9A), blocked C5b surface deposition (Figure 3E) and the inactive linear form showed no inhibitory effects ( Figure 3F). The effects of the untagged and tagged Compstatin were comparable in this assay.
  • Comp_SCR15-20 is functional active and prevents complement-mediated lysis of activator surfaces rE.
  • the inhibitory activity of the COMP_CFH 15-20 is comparable to untagged Compstatin.
  • CO MP_CFH 15-20 inhibits complement mediated lysis on non-activator surfaces
  • complement inhibitors as therapeutics for complement-mediated human diseases requires their activity on human cells, which act as non-activator surfaces. Therefore the action of the tagged COMP_CFH 15-20 was assayed in hemolytic assays using, i.e. sheep erythrocytes that represent non-activator surfaces together with complement active, Factor H depleted human serum (HS7CFH). Depletion of the fluid phase regulator Factor H results in complement mediated lysis of sheep erythrocytes. In this set up COMP_CFH15-20 prevented lysis of sheep erythrocytes and the effect was dose dependent (Figure 4A).
  • COMP_CFH15-20 prevents complement-mediated damage of living nucleated cells
  • COMP_CFH 15-20 and SCR15-20 bound to intact CHO and to intact HUVEC cells with comparable intensities ( Figure 5A, and Figure 7).
  • CHO cells that lack human membrane bound regulators represent a good model system to assay complement-mediated damage by NHS.
  • Complement mediated cell damage can be followed by challenging cells with NHS and then assaying the uptake of the non-fluorescent dye resazurin and its intracellular enzymatic conversion into the fluorescent dye resorufin.
  • CHO cells were challenged with NHS or with HSACFH (Figure 5B).
  • Factor H The protective effect of Factor H was confirmed by preincubating CHO cells with purified Factor H prior to addition of HSACFH. This treatment increased the cellu- lar metabolic activity and the effect was dose dependent. Factor H, used at 40 ⁇ g / ml, showed a protective effect as revealed by the enhanced metabolic activity of CHO cells (Figure 5C).
  • COM P_CFH15-20 prevents C5b deposition on the surface of CHO necrotic cells.
  • COMP_CFH15-20 also binds to necrotic cellular surfaces
  • binding of COMP_CFH15-20 and SCR15-20 to necrotic CHO and HUVEC cells was compared by flow cytometry. Both COMP_CFH 15-20 and SCR15-20 bound to necrotic CHO and to necrotic HUVEC cells with comparable intensities ( Figure 6A, and Figure 7).
  • Damaged cells such as necrotic or apoptotic cells express a reduced amount of membrane bound complement regulators. Therefore functionally active fluid phase regulators are necessary to prevent the deposition of active complement components and a following inflammatory process.
  • the complement activating effect of HSACFH was measured on the necrotic cell surface of CHO via flow cytometry. To this end, the deposition of the complement component C5b was characterized in the presence of COMP CFH15-20, SCR15-20, as well as the untagged active (4W9A) and inactive Compstatin. First, C5b deposition on necrotic CHO cells was compared after exposure of the cells to active NHS or to HSACFH ( Figure 6B).
  • the proof of principle has been shown by the chimeric compounds build of the bacterial complement inhibitors EfB, Sbi and SCIN in combination with SCR 1 8-20 of factor H.
  • the addition of the targeting Tag does not influence the regulatory activity of the complement inhibitor. It is preferred that binding of the chimeric proteins is effected in the presence of heparin and other carbohydrates present on the surface of the cells to be protected. Typically heparin as well as other factors being beneficial, like C3b, are present in the blood.

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Abstract

La présente invention concerne un polypeptide recombinant chimérique représentant un puissant inhibiteur de l'activation du complément. Ledit polypeptide recombinant est un polypeptide recombinant chimérique comprenant en cinq, trois ou n'importe quel autre nombre du fragment terminal C du facteur H du complément (CFH) comprenant au moins un, deux ou même cinq séquences consensus courtes répétées en C15 à C20 du facteur H ou d'une protéine CFHR liée à un inhibiteur du complément, permettant l'inhibition de l'activation du complément. Par ailleurs, l'invention concerne des molécules d'acide nucléique isolées codant pour le polypeptide chimérique, des vecteurs d'expression et des cellules, ainsi que des compositions pharmaceutiques les contenant. De plus, la présente invention concerne des méthodes permettant la prophylaxie ou le traitement de maladies, troubles ou conditions médiés par le complément, comme des maladies inflammatoires ou des maladies auto-immunes, ainsi que combattant la surcorrection de l'activation du complément chez un sujet, comprenant l'étape consistant à administrer le puissant inhibiteur selon la présente invention. De plus, la présente invention concerne l'utilisation de l'inhibiteur de la présente invention pour l'enrobage d'un quelconque biomatériau qui est exposé au sang, au sérum humain, ou à n'importe quel type de fluide corporel.
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Cited By (6)

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WO2014151919A1 (fr) * 2013-03-14 2014-09-25 Baxter International Inc. Facteur h pour transplantation
WO2015057893A3 (fr) * 2013-10-15 2015-06-25 The Texas A&M University System Compositions et utilisation d'un motif de liaison au fibrinogène en présence d'efb et de coa, pour vaccin contre le staphylococcus aureus et administration de médicament
US10155983B2 (en) 2014-03-31 2018-12-18 Machaon Diagnostics, Inc. Method of diagnosis of complement-mediated thrombotic microangiopathies
US11524050B2 (en) 2018-01-15 2022-12-13 Complement Therapeutics Limited C3B binding polypeptide
WO2023161662A1 (fr) * 2022-02-28 2023-08-31 Invizius Limited Composition de fluide de dialyse péritonéale comprenant un inhibiteur du complément
WO2025172139A1 (fr) 2024-02-14 2025-08-21 Christine Skerka Délétion de l'homologue de souris de fhr1 humain (fhre) qui protège les apoe-/-souris contre l'athérosclérose

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014151919A1 (fr) * 2013-03-14 2014-09-25 Baxter International Inc. Facteur h pour transplantation
AU2014236778B2 (en) * 2013-03-14 2019-01-03 Baxalta GmbH Factor H for transplantation
WO2015057893A3 (fr) * 2013-10-15 2015-06-25 The Texas A&M University System Compositions et utilisation d'un motif de liaison au fibrinogène en présence d'efb et de coa, pour vaccin contre le staphylococcus aureus et administration de médicament
US10155983B2 (en) 2014-03-31 2018-12-18 Machaon Diagnostics, Inc. Method of diagnosis of complement-mediated thrombotic microangiopathies
US11524050B2 (en) 2018-01-15 2022-12-13 Complement Therapeutics Limited C3B binding polypeptide
US12383600B2 (en) 2018-01-15 2025-08-12 Complement Therapeutics Limited C3B binding polypeptide
WO2023161662A1 (fr) * 2022-02-28 2023-08-31 Invizius Limited Composition de fluide de dialyse péritonéale comprenant un inhibiteur du complément
WO2025172139A1 (fr) 2024-02-14 2025-08-21 Christine Skerka Délétion de l'homologue de souris de fhr1 humain (fhre) qui protège les apoe-/-souris contre l'athérosclérose

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