EP3083664A2

EP3083664A2 - Recombinant proteins having factor h activity

Info

Publication number: EP3083664A2
Application number: EP14830995.8A
Authority: EP
Inventors: Toufik ABACHE
Original assignee: LFB SA
Current assignee: LFB SA
Priority date: 2013-12-20
Filing date: 2014-12-19
Publication date: 2016-10-26
Also published as: JP2017500049A; FR3015484A1; WO2015092335A2; US20170190753A1; CA2934558A1; JP2020014468A; WO2015092335A3

Abstract

The invention relates to a recombinant protein having factor H activity.

Description

The invention relates to a recombinant protein having an H factor activity. BACKGROUND OF THE INVENTION

Factor H is a 155 kDa plasma protein whose main function is the regulation of complement activity induced by the alternative pathway. Factor H is composed of 20 short repeat consensus (SCR) domains (also called CCP or SHUSHI) of about 60 amino acids linked together by a short linker sequence of 3 to 8 amino acids. The SCR1-4 domains carry the activity of accelerating the dissociation of C3 and C5 convertases and the factor I regulatory activity which allows the inactivation of the C3b protein. These N-terminal domains are sufficient to regulate the activity of C3 convertase in the liquid phase, but the SCR19-20 domains are necessary for the activity of factor H on the cell surface. The other Factor H SCR domains may also contribute more or less directly to the activity of the factor H, some containers of the binding sites for other molecules such as heparan sulfates and glycosaminoglycans, pentraxins (CRP, PTX3), fibromodulin or malondialdehyde. Some of these domains also contain glycosylation sites that may contribute to the half-life of the molecule and the efficiency of its production in recombinant form, others may have an important role for the three-dimensional conformation of factor H, as for example, SCR12, SCR13, SCR14 domains which confer on the H factor a particular hairpin-like folding (Schmidt et al., J. Mol Biol., 2010 January 08; 395 (1): 105-122).

Factor H is a promising therapeutic factor for the treatment of many diseases related to dense deposits of C3 or complement activation or uncontrolled inflammation. However, in some indications such as age-related macular degeneration (AMD), type 2 GNMP (Glomerulonephritis Membrano -Proliferate), atypical HUS (Uremic Hemorrhagic Syndrome), or certain autoimmune diseases, the use of Factor H could be limited due to some disadvantages: bioavailability, pharmacokinetics and pharmacodynamics, immunogenicity, heparan sulfate binding, binding to unidentified ligands, binding to pathogens allowing immune escape (S. pneumoniae, N.meningitidis, ...) and These disadvantages are related to the molecular properties of the factor H and thus to the organization of the SCR domains of the factor H.

A factor H derivative combining the CRI to SCR4 S domains with the S CRI 9 and SCR20 H factor domains (Hebecker et al, Journal of Immunology 2013, June 10, published online) has been proposed. In this factor H derivative constructed to direct the activity of factor H on the cell surface, the two domains of complement regulation and surface recognition are linked together by the 6 naturally occurring amino acids found between the fourth domain cysteine. SCR4 and the first cysteine of the SCR19 domain.

However, other domains of the factor H could also be necessary to obtain a therapeutic effectiveness superior to that of the factor H, such as for example to obtain a better bond with the cells, to preserve or to increase the half-life of the FH or to preserve or favor the characteristic folding of the FH hairpin. There is therefore a need for molecules derived from factor H, said factor H-derived molecules having a factor H activity, having an advantage over factor H or not having the disadvantages of factor H, and whose activity would be preference retained or improved over that of factor H.

Summary of the invention

The Applicant here responds to this need by proposing recombinant proteins derived from factor H having a rearrangement of the number and organization of SCR domains of factor H which retain an activity of factor H. The recombinant proteins according to the invention comprise at least one the SCR1-4 domains (SCR1, SCR2, SCR3 and SCR4, in that order), SCR19 and SCR20 of the H factor.

An object of the invention therefore relates to a recombinant protein having a factor H activity, comprising, from the N-terminus to the C-terminus, a first amino acid sequence comprising at least the domains SCR1 to SCR4 of factor H, and a second amino acid sequence comprising at least the domains SCR19 and SCR20 of factor H, said recombinant protein not being a natural factor H, and provided that if the first sequence consists of domains SCR1 to SCR4 and the second sequence consists of domains SCR19 and SCR20, the linker is a synthetic linker.

The subject of the invention is also a nucleic acid encoding a recombinant protein according to the invention, a vector comprising such a nucleic acid, a host cell comprising such a nucleic acid or such a vector and a transgenic animal whose genome comprises such a nucleic acid.

The invention also relates to a recombinant protein according to the invention, for the treatment of diseases due to uncontrolled inflammation or deposition of uncontrolled C3 convertase. More generally, the recombinant protein according to the invention may be useful for the treatment of any disease for which anti-complement factor H activity is beneficial.

Detailed description of the invention

In one embodiment, the present invention relates to a factor H-derived recombinant protein having a rearrangement of the SCR domains, both in terms of their number and their organization, said recombinant protein comprising, in this order, a first sequence of acids. amino acids comprising the H-factor SCR1-4 domains and a second amino acid sequence comprising the F-factor SCR19 and SCR20 domains. The first and / or second amino acid sequence may further comprise one or more other factor SCR domains. H rearranged

In one embodiment, one or more SCR domains of the factor H may be present in multiple copies. For example, a recombinant protein according to the invention may include one, two, three, or more than three copies of the same SCR, for example one, two, three or more than three copies of the domain SCR1, SCR2, SCR3, SCR4 , SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR11, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and / SCR20 of the factor H. According to a particular embodiment, the recombinant protein according to The invention comprises one, two, three or more than three copies of the SCR7 domain of factor H, in particular the SCR7 domain of the Y402 variant of factor H. Another variant includes a recombinant protein comprising multiple copies of a set of SCR domains. For example, the recombinant protein according to the invention may comprise one, two, three or more repetitions of SCR1 to SCR4 domains, i.e. the protein comprises the (SCR1-SCR4) _n motif, n being an integer equal to 1, 2, 3 or greater than 3, in particular n being equal to 1, 2 or 3. In another embodiment, the domain or set of domains present in multiple copies are not repetitions domains or set of domains, but may be present in the recombinant protein separated by other SCR domains. In other words, the invention relates in particular to recombinant proteins comprising, in this order, the domains SCR1-SCR4, SCR7, SCR7 and SCR19-SCR20, or the SCR1-SCR4, SCR7, SCR1-SCR4, and SCR19-20 domains, or any other possible combination of these domains.

The factor H from which the protein according to the invention is derived can be any factor H whose activity is that of the natural factor H. In particular, "H factor" is understood to mean any protein having the amino acid sequence of human native H factor or from another species (for example bovine, porcine, canine or murine). Preferentially, the recombinant protein is derived from human factor H. The term also includes any recombinant, derivative or mutant having a sequence substantially homologous to native H-factor. The term "substantially homologous sequence" includes any sequence subject to one or more substitutions, additions and / or deletions, preferably conservative. The terms "conservative substitutions, additions and / or deletions" express any replacement, addition or deletion of amino acid residue by another, without major alteration of the general conformation and / or biological activity of factor H. Conservative substitution includes , but not limited to, replacement with an amino acid having similar properties (such as, for example, shape, polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity and the like). Amino acids with similar properties are well known in the art. The term "H factor" further includes natural allelic variations and / or factor H isoforms found naturally in individuals of the same species, and any form or degree of glycosylation or other post-translational modification. Also included in the term "H-factor" are homologues or derivatives of factor H which exhibit the same activity, or a higher biological activity with respect to the activity of the wild-type form and / or which have a sequence identity of from at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%.

The factor H variant used to design the recombinant protein of the present invention may in particular be a variant mentioned in the website http://www.uniprot.org/uniprot/P08603.

The anti-complement activity of factor H is reflected in the regulation of the alternative complement pathway by maintaining a basal level of C3b molecule. Factor H complies with factor B for C3b binding and accelerates the dissociation of the already formed alternating C3 convertase (C3bBb). It acts as a cofactor of Factor I in the proteolysis of C3b, free or bound to the cell surface, which leads to the inactive form C3bi. Thus, the immune complexes composed of an antigen-antibody complex associated with the complement component C3b or the activating factors of the alternative complement pathway (bacterial surfaces, infected cells, yeasts, parasites, lipopolysaccharides, endotoxins) can no longer activate the subsequent cascade of complement (C5-C9 components). The term "biological activity" of factor H thus includes here the ability to inhibit C3 convertase and / or to act as a factor I co-factor, resulting in the inhibition of activation of the complement cascade. In a particular embodiment, the recombinant protein retains the biological activity of a plasma human factor H.

The biological activity of human plasma H-factor includes regulation of Factor I activity, inhibition of alternating C3 convertase formation and acceleration of C3 convertase dissociation.

The methods for determining the biological activities above are known to those skilled in the art.

In a more particular embodiment, said recombinant protein retains the biological activity of a plasma human factor H to accelerate the dissociation of C3 convertase. The amount of dissociated C3 convertase can be determined as a function of the concentration of recombinant protein added. The IC50 is determined from the equation calculated for a variable slope sigmoid.

In another more particular embodiment, said recombinant protein retains the biological activity of a plasma H factor to regulate factor I activity.

Furthermore, the biological activity of factor H can be evaluated by measuring the protection activity of red blood cells against lysis by complement according to procedures well known in the state of the art.

The sequence SEQ ID NO: 1 represents the amino acid sequence of the Y402 variant of human factor H in which the amino acid at position 402 is a tyrosine. This sequence does not include a signal peptide. The sequence SEQ ID NO: 2 represents the amino acid sequence of the human factor H402 variant in which the amino acid at position 402 is a histidine. This sequence does not include a signal peptide. In one embodiment, one or more SCR domains of the recombinant protein according to the invention are derived from one or the other of these two variants. According to one particular embodiment, the recombinant protein comprises the SCR7 domain of the Y402 variant of the factor H. According to variants of this embodiment, the first sequence of the recombinant protein comprises the S domains CRI to SCR7, the S domains CRI to SCR8 or S domains CRI to SCR9 of the factor H, the domain SCR7 in this first sequence being the domain SCR7 of the variant Y402 of the factor H. In these variant embodiments, the other domains SCR can be derived from variant Y402 of factor H or another variant of factor H. Moreover, the first sequence can in particular be combined with a second amino acid sequence comprising domains SCR19 and SCR20, SCR18 to SCR20, SCR17 to SCR20 or SCR16 to SCR20 of factor H, the second amino acid sequence being in particular derived from the Y402 variant or the H402 factor H variant. These recombinant proteins include the Y402 polymorphism present in SCR7.

In one embodiment, the recombinant protein according to the invention comprises, in this order, the domains SCR1, SCR2, SCR3, SCR4, SCR19 and SCR20 of the factor H. In this embodiment, the domains SCR4 and SCR19 are linked between them by an unnatural (or synthetic) linker consisting of a sequence comprising between 2 and 20 amino acids. The term "non-natural (or synthetic) linker" refers in particular to a linker that does not correspond to the sequence found between the fourth cysteine of the SCR4 domain and the first cysteine of the SCR19 domain. In particular, the amino acid sequence of the linker is chosen such that it is encoded by a nucleic acid which, when introduced into a cloning and / or expression vector, comprises a unique restriction site ( see below). For example, the linker may be of GASG sequence (SEQ ID NO: 3).

According to one embodiment, the first or second amino acid sequence of the recombinant protein according to the invention comprises one or more additional SCR domains of the factor H chosen from among the domains SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR11, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17 and SCR18. The order of these domains in the recombinant protein may correspond to the order of the same domains in the natural factor H. Alternatively, the order of the domains may be different from that of the natural H factor.

The amino acid sequence of the SCR1 domain (amino acids 21 to 80) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 4 (CNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPL R C). The sequence of the SCR1 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, especially at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 4. In one embodiment, the sequence of the SCR1 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 4.

The amino acid sequence of the SCR2 domain (amino acids 85 to 141) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 5 (CGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPIC ). The sequence of the SCR2 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99% identity with the sequence SEQ ID NO: 5. In one embodiment, the sequence of the SCR2 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 5.

The amino acid sequence of the SCR3 domain (amino acids 146 to 205) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 6 (CLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKE KPKC). The sequence of the SCR3 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 6. In one embodiment, the sequence of the SCR3 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 6.

The amino acid sequence of the SCR4 domain (amino acids 210 to 262) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 7 (CKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSC). The sequence of the SCR4 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99% identity with the sequence SEQ ID NO: 7. In one embodiment, the sequence of the SCR4 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 7.

The amino acid sequence of the SCR5 domain (amino acids 266 to 320) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 8 (CDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRC). The sequence of the SCR5 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 8. In one embodiment, the sequence of the SCR5 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 8.

The amino acid sequence of the SCR6 domain (amino acids 325 to 385) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 9 (CDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGW SPAVPC). The sequence of the SCR6 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99%. % identity with the sequence SEQ ID NO: 9. In one embodiment, the sequence of the SCR6 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 9.

The amino acid sequence of the SCR7 domain (amino acids 389 to 442) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 10 (CYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRC). The sequence of the SCR7 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, especially at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 10. embodiment, the sequence of the SCR7 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 10.

The amino acid sequence of the SCR8 domain (amino acids 448 to 505) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 11 (CSKSSIDIENGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTC). The sequence of the SCR8 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 1. one embodiment, the sequence of the SCR8 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 11.

The amino acid sequence of the SCR9 domain (amino acids 509 to 564) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 12 (CDIPVFMNARTKNDFTWFKLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPIC). The sequence of the SCR9 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 12. embodiment, the sequence of the SCR9 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 12.

The amino acid sequence of the SCR10 domain (amino acids 569 to 623) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 13 (CELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPIC). The sequence of the SCR10 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 13. embodiment, the sequence of the domain SCR10 introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 13.

The amino acid sequence of the SCR11 domain (amino acids 630 to 674) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 14 (CGPPPELLNGNVKEKTKEEYGHSEVVEYYCNPRFLMKGPNKIQCVDGEWTTLPVC). The sequence of the SCR11 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 14. embodiment, the sequence of the SCR11 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 14.

The amino acid sequence of the SCR12 domain (amino acids 691 to 744) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 15 (CGDIPELEHGWAQLSSPPYYYGDSVEFNCSESFTMIGHRSITCIHGVWTQLPQC). The sequence of the SCR12 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 15. embodiment, the sequence of the SCR12 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 15.

The amino acid sequence of the SCR13 domain (amino acids 753 to 803) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 16 (CKSSNLIILEEHLKNKKEFDHNSNIRYRCRGKEGWIHTVCINGRWDPEVNC). The sequence of the SCR13 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 16. embodiment, the sequence of the SCR13 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 16.

The amino acid sequence of domain SCR14 (amino acids 811 to 864) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 17 (CPPPPQIPNSHNMTTTLNYRDGEKVSVLCQENYLIQEGEEITCKDGRWQSIPLC). The sequence of the SCR14 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 17. embodiment, the sequence of the SCR14 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 17. The amino acid sequence of domain SCR15 (amino acids 869 to 926) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 18 (CSQPPQIEHGTINSSRSSQESYAHGTKLSYTCEGGFRISEENETTCYMGKWSSPPQC). The sequence of the SCR15 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 18. embodiment, the sequence of the SCR15 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 18.

The amino acid sequence of domain SCR16 (amino acids 931 to 984) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 19 (CKSPPEISHGVVAHMSDSYQYGEEVTYKCFEGFGIDGPAIAKCLGEKWSHPPSC). The sequence of the SCR16 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 19. embodiment, the sequence of the SCR16 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 19.

The amino acid sequence of domain SCR17 (amino acids 989 to 1043) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 20 (CLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNVTCINSRWTGRPTC). The sequence of the SCR17 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99% identity with the sequence SEQ ID NO: 20. In one embodiment, the sequence of the SCR17 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 20.

The amino acid sequence of the SCR18 domain (amino acids 1048 to 1102) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 21 (CVNPPTVQNAYIVSRQMSKYPSGERVRYQCRSPYEMFGDEEVMCLNGNWTEPPQC). The sequence of the SCR18 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, especially at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 21. In one embodiment, the sequence of the SCR18 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 21.

The amino acid sequence of domain SCR19 (amino acids 1109 to 1163) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 22 (CGPPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCR GQWSEPPKC). The sequence of the SCR19 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 22. In one embodiment, the sequence of the SCR18 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 22.

The amino acid sequence of domain SCR20 (amino acids 1167 to 1231) of human factor H variant Y402 is represented by the sequence SEQ ID NO: 23 (CVISREIMENYNIALRWTAKQKLYSRTGESVEFVCKRGYRLSSRSHTLRTTCWDGKL EYPTCAKR). The sequence of the SCR18 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 23. In one embodiment, the sequence of the SCR20 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 23.

In one embodiment, the first or second amino acid sequence comprises at least the SCR12, SCR13, and SCR14 domains of the H factor. In another embodiment, none of the SCR12, S CRI3, and S CRI4 domains are is present in the recombinant protein according to the invention. An embodiment variant of the present invention relates to a recombinant protein whose SCR domains consist, in this order, of domains SCR1, SCR2, SCR3, SCR4, SCR12, SCR13, SCR14, SCR19 and SCR20. Such a protein is represented in the sequence SEQ ID NO: 142.

Each SCR domain included in the first or second amino acid sequence may be linked to the contiguous SCR (s) by means of the natural linker found between said SCR domains, as appropriate. Alternatively, the linker between each SCR domain of the recombinant protein may be a non-natural linker. The unnatural linker may consist of a sequence comprising between 2 and 20 amino acids, in particular between 3 and 8 amino acids. Moreover, the link between the first and the second amino acid sequence can be achieved by means of a natural or synthetic linker present in the C-terminal position of the first amino acid sequence or in the N-terminal position of the second sequence. in amino acids. For example, the first and second polypeptide may be linked by a GASG sequence linker (SEQ ID NO: 3). Furthermore, the first and second amino acid sequences may be linked together by a linker combining the natural linker sequence according to the last domain of the first sequence (ie the C-terminal domain of the first sequence) and a linker. synthetic such as the linker GASG. The Natural linkers found at the C-terminal position of each of the SCR domains of the factor H are, for example: QKRP (SEQ ID NO: 143) for the SCR1 domain; EVVK (SEQ ID NO: 144) for the SCR2 domain; VEIS (SEQ ID NO: 145) for the SCR3 domain; EEKS (SEQ ID NO: 146) for the SCR4 domain; TLKP (SEQ ID NO: 147) for the SCR5 domain; LRK for the SCR6 domain; IRVKT (SEQ ID NO: 148) for the SCR7 domain; IKS for the SCR8 domain; YERE (SEQ ID NO: 149) for the SCR9 domain; KEQVQS (SEQ ID NO: 150) for the SCR10 domain; IVEEST (SEQ ID NO: 151) for the SCR1 domain l; VAIDKLKK (SEQ ID NO: 152) for the SCR12 domain; SMAQIQL (SEQ ID NO: 153) for the domain SCR13; VEKIP (SEQ ID NO: 154) for the SCR14 domain; EGLP (SEQ ID NO: 155) for the SCR15 domain; IKTD (SEQ ID NO: 156) for the SCR16 domain; RDTS (SEQ ID NO: 157) for the SCR17 domain; KDSTGK (SEQ ID NO: 158) for the SCR18 domain; LHP for the SCR19 domain.

According to one embodiment, the first amino acid sequence comprises the domains SCR1, SCR2, SCR3 and SCR4 of the factor H.

According to one embodiment, the first amino acid sequence comprises the domains SCR1, SCR2, SCR3, SCR4 and SCR5 of the factor H.

According to another embodiment, the first amino acid sequence comprises the domains SCR1, SCR2, SCR3, SCR4, SCR5 and SCR6 of the factor H.

According to another embodiment, the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6 and SCR7 domains of the factor H.

According to another embodiment, the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7 and SCR8 domains of the H factor.

According to another embodiment, the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8 and SCR9 domains of the H factor.

According to another embodiment, the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9 and SCR10 domains of the H factor.

According to another embodiment, the first amino acid sequence comprises the domains SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9, SCR10 and SCR1, factor H.

According to another embodiment, the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR1 and SCR12 H-factor domains. According to another embodiment, the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR1, SCR12 and SCR13 H-factor domains.

According to one embodiment, the second amino acid sequence comprises the domains SCR19 and SCR20 of the factor H.

According to one embodiment, the second amino acid sequence comprises the domains SCR18, SCR19 and SCR20 of the factor H.

According to one embodiment, the second amino acid sequence comprises the domains SCR17, SCR18, SCR19 and SCR20 of the factor H.

According to one embodiment, the second amino acid sequence comprises the domains SCR16, SCR17, SCR18, SCR19 and SCR20 of the factor H.

According to one embodiment, the second amino acid sequence comprises the domains SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 of the factor H.

According to one embodiment, the second amino acid sequence comprises the domains

SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 of factor H.

SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 of factor H.

SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 of factor H.

SCR1, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 of factor H.

According to one embodiment, the second amino acid sequence comprises the SCR10, SCR1, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 domains of the H factor.

According to one embodiment, the second amino acid sequence comprises the SCR9, SCR10, SCR1, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 domains of the H factor.

According to one embodiment, the second amino acid sequence comprises the SCR8, SCR9, SCR10, SCR1, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 domains of the H factor.

According to another embodiment, the recombinant protein having a factor H activity comprises the first and second amino acid sequences listed in Table 1 below. below, these sequences being separated by a linker, in particular an artificial linker, in particular the GASG linker (SEQ ID NO: 3).

Table 1

First sequence Second sequence First sequence Second sequence

SCR1 to SCR11 SCR15 to SCR20 SCR1 to SCR12 SCR14 to SCR20

SCR1 to SCR11 SCR14 to SCR20 SCR1 to SCR12 SCR13 to SCR20

SCR1 to SCR11 SCR13 to SCR20 SCR1 to SCR13 SCR19 to SCR20

SCR1 to SCR11 SCR12 to SCR20 SCR1 to SCR13 SCR18 to SCR20

SCR1 to SCR12 SCR19 to SCR20 SCR1 to SCR13 SCR17 to SCR20

SCR1 to SCR12 SCR18 to SCR20 SCR1 to SCR13 SCR16 to SCR20

SCR1 to SCR12 SCR17 to SCR20 SCR1 to SCR13 SCR15 to SCR20

SCR1 to SCR12 SCR16 to SCR20 SCR1 to SCR13 SCR14 to SCR20

SCR1 to SCR12 SCR15 to SCR20

According to a particular embodiment, the recombinant protein having a factor H activity comprises the first and second amino acid sequences listed in Table 2 below, these sequences being separated by a linker, in particular an artificial linker, in particular the linker G AS G (SEQ ID NO: 3).

Table 2

First sequence Second sequence

SCR1 to SCR4 SCR19 to SCR20

SCR1 to SCR4 SCR16 to SCR20

SCR1 to SCR4 SCR15 to SCR20

SCR1 to SCR4 SCR14 to SCR20

SCR1 to SCR4 SCR11 to SCR20

SCR1 to SCR4 SCR10 to SCR20

SCR1 to SCR4 SCR9 to SCR20

SCR1 to SCR4 SCR8 to SCR20

SCR1 to SCR7 SCR16 to SCR20

SCR1 to SCR7 SCR15 to SCR20

SCR1 to SCR7 SCR14 to SCR20

SCR1 to SCR7 SCR11 to SCR20

SCR1 to SCR7 SCR10 to SCR20

SCR1 to SCR7 SCR9 to SCR20

SCR1 to SCR7 SCR8 to SCR20

SCR1 to SCR8 SCR16 to SCR20 According to a preferred variant of the invention, the first sequence comprises the domains SCR1 to SCR7 of factor H, and the second sequence is chosen from the group consisting of: domains SCR9 to SCR20,

domains SCR11 to SCR20; and

domains SCR14 to SCR20 of the factor H.

According to another preferred variant, the first sequence comprises the domains SCR1 to SCR8 and the second sequence comprises the domains SCR10 to SCR20.

According to another preferred variant, the first sequence comprises the domains SCR1 to SCR4 of the factor H and the second sequence comprises the domains SCR16 to SCR20 of the factor H, a third sequence comprising the domain SCR7 of the factor 7 being between the first and the second sequence, and being more particularly separated from these by linkers such as those described above.

According to one embodiment, the first amino acid sequence comprises a signal peptide (PS) in the N-terminal position. The signal peptide can be the natural signal peptide of factor H (MRLLAKIICLMLWAICVA - SEQ ID NO: 24), the signal peptide of a protein different from factor H, or a signal peptide described in application PCT / 2001/050544, especially the peptide MRWSWIFLLLLSITSANA (SEQ ID NO: 25, or otherwise referred to as PS-MB7 thereafter). The natural signal peptide of a protein different from human factor H may be a signal peptide chosen from the signal peptides of all the proteins which are secreted in eukaryotes and in particular in mammals and more particularly in humans, such as those of immunoglobulins. growth factors such as ΓΕΡΟ, hormones such as insulin, enzymes such as trypsinogen, coagulation factors such as prothrombin. The presence of a signal peptide makes it possible to confer a better secretion of recombinant protein in the culture medium.

According to one embodiment, the invention relates to one of the peptides of Table 1 in which the first amino acid sequence comprises at the N-terminal such a signal peptide, in particular the natural peptide of the factor H of sequence SEQ ID NO: 24 or the signal peptide PS-MB7 of sequence SEQ ID NO: 25. According to another embodiment, the recombinant protein according to the invention is chosen from one of the proteins of Table 1, the first and second sequences being separated by a linker, including a GASG linker (SEQ ID NO: 3).

According to one particular embodiment, the recombinant protein according to the invention is chosen from one of the proteins listed in Table 2 below in which the first amino acid sequence comprises a signal peptide of sequence SEQ ID NO: 25 and the first and second sequences are separated by a GASG sequence linker (SEQ ID NO: 3).

Table 3

SEQ ID First linker sequence Second sequence

NO:

57 PS-MB7 + SCR1 to SCR9 GASG SCR15 to SCR20

58 PS-MB7 + SCR1 to SCR9 GASG SCR1 4 to SCR20

59 PS-MB7 + SCR1 to SCR9 GASG SCR1 3 to SCR20

60 PS-MB7 + SCR1 to SCR9 GASG SCR1 2 to SCR20

61 PS-MB7 + SCR1 to SCR9 GASG SCR1 1 to SCR20

62 PS-MB7 + SCR1 to SCR9 GASG SCR10 to SCR20

63 PS-MB7 + SCR1 to SCR1 0 GASG SCR19 to SCR20

64 PS-MB7 + SCR1 to SCR1 0 GASG SCR16 to SCR20

65 PS-MB7 + SCR1 to SCR1 0 GASG SCR15 to SCR20

66 PS-MB7 + SCR1 to SCR1 0 GASG SCR1 4 to SCR20

67 PS-MB7 + SCR1 to SCR1 0 GASG SCR1 3 to SCR20

68 PS-MB7 + SCR1 to SCR1 0 GASG SCR1 2 to SCR20

69 PS-MB7 + SCR1 to SCR1 0 GASG SCR1 1 to SCR20

70 PS-MB7 + SCR1 to SCR1 1 GASG SCR19 to SCR20

71 PS-MB7 + SCR1 to SCR1 1 GASG SCR16 to SCR20

72 PS-MB7 + SCR1 to SCR1 1 GASG SCR15 to SCR20

73 PS-MB7 + SCR1 to SCR1 1 GASG SCR1 4 to SCR20

74 PS-MB7 + SCR1 to SCR1 1 GASG SCR1 3 to SCR20

75 PS-MB7 + SCR1 to SCR1 1 GASG SCR1 2 to SCR20

76 PS-MB7 + SCR1 to SCR12 GASG SCR19 to SCR20

77 PS-MB7 + SCR1 to SCR12 GASG SCR16 to SCR20

78 PS-MB7 + SCR1 to SCR12 GASG SCR15 to SCR20

79 PS-MB7 + SCR1 to SCR12 GASG SCR1 4 to SCR20

80 PS-MB7 + SCR1 to SCR12 GASG SCRl 3 to SCR20

81 PS-MB7 + SCR1 to SCR1 3 GASG SCR19 to SCR20

82 PS-MB7 + SCR1 to SCR1 3 GASG SCR16 to SCR20

83 PS-MB7 + SCR1 to SCR1 3 GASG SCR15 to SCR20

84 PS-MB7 + SCR1 to SCR1 3 GASG SCR1 4 to SCR20

According to a particular embodiment, the recombinant protein according to the invention contains a first sequence comprising the domains SCR1 to SCR4 of factor H, and a second sequence chosen from the group consisting of:

domains SCR1 6 to SCR20,

domains SCR1 to SCR20;

- domains SCR10 to SCR20; and

domains SCR8 to SCR20 of the factor H.

According to another embodiment, the recombinant protein according to the invention contains a first sequence comprising the domains SCR1 to SCR7 of factor H, and a second sequence comprising the SCR1 6 to SCR20 domains of the factor H, said second sequence preferably being chosen from the group consisting of:

- domains SCRl 5 to SCR20

domains SCR1 4 to SCR20;

domains SCR1 1 to SCR20;

- domains SCR10 to SCR20;

- domains SCR9 to SCR20; and

- Domain SCR8 to SCR20 of the factor H. According to another variant, the invention relates to a recombinant protein as described above, the first sequence comprising the domains SCR1 to SCR8 of the factor H, and the second sequence comprising the domains SCR1 6 to SCR20 of the factor H, said second sequence preferably comprising the domains SCR10 to SCR20 of the factor H. According to another embodiment, the recombinant protein according to the invention contains a first sequence comprising the domains SCR1 to SCR4 of the factor H and a second sequence comprising the domains SCR1 6 to SCR20 of the factor H, a third sequence comprising the domain SCR7 of the factor H being between the first and the second sequence.

According to one variant of the invention, the first sequence comprises a signal peptide of sequence SEQ ID NO: 25 and domains SCR1 to SCR7 of factor H, and the second sequence is chosen from the group consisting of:

- domains SCR9 to SCR20,

domains SCR1 1 to SCR20; and

domains SCR1 4 to SCR20 of the factor H.

In a particular embodiment of this variant, the first and the second sequences are separated by a GASG sequence linker. According to another preferred variant, the first sequence comprises a signal peptide of sequence SEQ ID NO: 25 and domains SCR1 to SCR8 and the second sequence comprises domains SCR10 to SCR20. In a particular embodiment of this variant, the first and the second sequences are separated by a GASG sequence linker. According to another preferred variant, the first sequence comprises a signal peptide of sequence SEQ ID NO: 25 and domains SCR1 to SCR4 of factor H and the second sequence comprises domains SCR16 to SCR20 of factor H, a third sequence comprising domain SCR7 factor 7 being between the first and the second sequence, and being separated therefrom by linkers such as those described above. In a particular embodiment of this variant, the first and the third sequences, and the third and the second sequences are separated by a GASG sequence linker. Such a sequence is represented in SEQ ID NO: 159. The present invention also relates to a pharmaceutical composition comprising a recombinant protein according to the invention.

The invention also relates to a nucleic acid construct encoding a recombinant protein having a factor H activity as described above.

Advantageously, the nucleic acids encoding the recombinant proteins according to the invention have undergone codon optimization.

Codon optimization aims to replace natural codons by codons whose transfer RNA (tRNA) carrying the amino acids are most common in the cell type considered. The mobilization of frequently encountered tRNAs has the major advantage of increasing the translation speed of the messenger RNAs (mRNA) and therefore of increasing the final titre (JM Carton et al., Protein Expr Purif, 2007). Sequence optimization also plays on the prediction of mRNA secondary structures that could slow down reading by the ribosomal complex. Sequence optimization also has an impact on the percentage of G / C that is directly related to the half-life of the mRNAs and therefore to their potential to be translated (Chechetkin, J. of Theoretical Biology 242, 2006 922-934 ).

Codon optimization can be done by substitution of natural codons using codon frequency (Codon Usage Table) tables for mammals and more specifically for Homo sapiens. There are algorithms available on the internet and made available by the suppliers of synthetic genes (DNA2.0, GeneArt, MWG, Genscript) that make this sequence optimization possible.

By way of illustration, a codon-optimized sequence is represented by the sequence SEQ ID NO: 85. This sequence comprises the natural signal peptide of the factor H.

The nucleic acids according to the invention may comprise a unique restriction site between the two nucleic acid sequences encoding the first and the second acid sequence. amines of the recombinant protein according to the invention. This unique restriction site may especially correspond to the Nhe I site present in the portion encoding the GASG linker (nucleic sequence: GCCGCTAGCGCC (SEQ ID NO: 86), the underlined portion corresponding to the NheI site which corresponds to the amino acids AS mentioned above. This unique restriction site makes it possible to envisage an improvement in the recombinant proteins produced, in particular by facilitated introduction of one or more additional SCR domains at the level of the protein sequence corresponding to said restriction site, or by introduction of a sequence into Amino acids other than an SCR domain may be, for example, another protein domain not belonging to the natural factor H, or a linker of different size and sequence (especially a longer linker).

The nucleic acids encoding the recombinant protein according to the invention may be constructed according to any method known to those skilled in the field of molecular biology. Advantageously, however, said nucleic acid is constructed in two stages according to a method peculiar to the present invention. For example, a nucleic acid library cloned into cloning or expression vectors may be constructed. Each vector of the library contains a sequence encoding one of the first or second amino acid sequences comprising the recombinant protein according to the invention. Thus, a vector comprising the sequences coding a first amino acid sequence comprising at least the sequences of the SCR1 to SCR4 domains of factor H (or N-Ter vector) is described. Similarly, there is described a vector comprising the sequences encoding a second amino acid sequence comprising at least the sequences of the domains SCR19 and SCR20 of the factor H (or C-Ter vector).

The N-Ter and C-Ter vectors are designed to include unique restriction sites useful for excising and then assembling nucleic acid fragments encoding each of the portions of the recombinant protein into a single vector. The constructs allowing the expression of the proteins of Table 2 above can thus be produced from 8 N-Ter vectors and 10 C-Ter vectors. This strategy is described in the examples below.

The nucleic acids according to the invention may also comprise any useful sequence, in particular any sequence which makes it possible to optimize the expression or the secretion of the recombinant protein or to facilitate the cloning and subcloning of the nucleic acids of the invention. For example, the nucleic acid may comprise at least one restriction site, a coding sequence and / or a signal peptide coding sequence. In 3 ', it may in particular be useful to introduce a sequence encoding an amino acid unit useful for the labeling or purification of the recombinant protein, for example a histidine tag, and one or more restriction sites.

In one embodiment, the nucleic acid construct according to the invention comprises a sequence encoding a signal peptide chosen from:

a nucleic acid represented by the sequence SEQ ID NO: 87 and coding the natural signal peptide of the factor H, or

a nucleic acid represented by the sequence SEQ ID NO: 88 or by a sequence having at least 85%, in particular 90%, especially 95% of sequence identity with the sequence SEQ ID NO: 88, and coding the signal peptide factor H, (optimized natural PS) or

a nucleic acid encoding a natural signal peptide of a protein different from the factor H, or

a nucleic acid encoding the signal peptide encoded by the sequence SEQ ID NO: 89 (described in application PCT / FR2011 / 050544) or by a sequence having at least 85%, in particular 90%), particularly 95% identity of sequence with the sequence SEQ ID NO: 89.

The sequence SEQ ID NO: 88 or a sequence having at least 85%, especially 90%>, especially 95% of sequence identity with the sequence SEQ ID NO: 88 is a sequence obtained by codon optimization from the sequence SEQ ID NO: 87.

The nucleic acid represented by the sequence SEQ ID NO: 89 or by a sequence having at least 85%, in particular 90%, especially 95% of sequence identity with the sequence SEQ ID NO: 89, encodes the artificial PS-signal peptide. MB7 described above.

In one embodiment, the nucleic acid construct encoding the recombinant protein according to the invention comprises, or consists of:

a nucleic acid encoding a signal peptide, in particular a nucleic acid represented by the sequence SEQ ID NO: 87 and encoding the natural signal peptide of factor H, or a nucleic acid represented by the sequence SEQ ID NO: 88 or by a sequence having at least 85%), especially 90%, particularly 95% of sequence identity with the sequence SEQ ID NO: 88, and coding the signal peptide of factor H, (optimized natural PS) or a nucleic acid encoding a natural signal peptide a protein different from the factor H, or a nucleic acid encoding the signal peptide encoded by the sequence SEQ ID NO: 89 (described in application PCT / FR2011 / 050544) or by a sequence having at least 85%, in particular 90% especially 95% sequence identity with the sequence SEQ ID NO: 89;

a nucleic acid encoding at least the domains SCR1, SCR2, SCR3 and SCR4 of factor H; a nucleic acid encoding a linker, in particular a GASG linker (SEQ ID NO: 3), said nucleic acid encoding a linker comprising a unique restriction site, in particular an NheI site;

a nucleic acid encoding at least the domains SCR19 and SCR20 of the factor H.

According to one embodiment, the nucleic acid construct allows the expression of a recombinant protein selected from one of the sequences SEQ ID NO: 26 to SEQ ID NO: 84. The invention also relates to an expression vector comprising the nucleic acid construct described above, operably linked to sequences for controlling the expression of said nucleic acid. The control sequences may in particular comprise a promoter (in particular a CMV promoter), an enhancer, and any sequence known to those skilled in the art useful for allowing the expression of the recombinant protein in a eukaryotic cell, in particular a mammalian cell.

In this respect, the invention also relates to a recombinant eukaryotic cell, in particular a mammalian cell, more particularly a non-human (eg a CHO cell) or human cell, transformed by means of the nucleic acid construct or the vector. expression according to the invention. In an advantageous embodiment of the present invention, the recombinant protein as described above is produced in the PER.C6® cell line or a HEK cell line, in particular the HEK 293F cell line. The PER.C6® cell line is derived from human primary retinal cells in which an Ad5 adenoviral DNA fragment that contains both the E1A gene and the ElB gene is inserted into the cells by a vector. This adenoviral DNA fragment makes it possible to impart immortality to the cells in which it is inserted, via the ElB protein which inhibits the p53 protein. The El A protein has a tropism for the hCMV viral promoter and allows its transactivation and the potentiation of the gene sequence which will be inserted 3 'of the latter and which may be the recombinant protein according to the invention.

The present invention particularly relates to the use of the cell line PER.C6® for the implementation of a method for preparing a recombinant protein having a factor H activity, in particular one of the proteins of sequence SEQ ID NO: 26 to SEQ ID NO: 84.

The present invention also particularly relates to the use of the HEK 293F cell line for implementing a method for preparing a recombinant protein according to the invention, in particular one of the recombinant proteins represented by one of the SEQ sequences. ID NO: 26 to SEQ ID NO: 84. The invention also relates to a process for the production of a recombinant protein having an activity of factor H. The method according to the invention, in particular one of the proteins represented by the sequences SEQ ID NO: 26 to 84, said method comprising the culture of a recombinant cell according to the invention transformed by a vector comprising the nucleic acid construct according to the invention coding for said recombinant protein.

The vector comprising such a nucleic acid may be any expression vector for eukaryotic cell lines known to those skilled in the art.

The transformation of the cell line can be carried out by electroporation, AMAXA type nucleofection, a "gene gun" (gun gene) or else using a transfection agent known to man of the art, such as cationic agents, liposomes or polymers such as fectin or PEI agent.

In a particular embodiment, the method according to the present invention comprises the following steps:

(i) transfection of a eukaryotic cell with an expression vector comprising a nucleic acid construct encoding the recombinant protein, to obtain a transfected cell,

(ii) culturing said transfected cell to obtain expression of the recombinant protein in the culture medium.

The expression vector may contain an antibiotic resistance gene to allow selection of transfected cells upon establishment of cells stably producing the protein of interest.

In one embodiment, the recombinant protein according to the invention is produced at a concentration greater than or equal to 10 mg / L as detected by ELISA assay of culture supernatant, after 7 days of production in batch mode.

The purification of the recombinant protein can be carried out by chromatographic techniques in one, two or more steps. The one-step purification may be an ion exchange column or an affinity column (heparin, factor H ligand or anti-factor H antibody). The purification in two steps may be a cation exchange column chromatography step followed by an anion exchange column chromatography step or an anion exchange column chromatography step followed by a column chromatography step cation exchange or an ion exchange column chromatography step followed by an affinity column chromatography step or an affinity column chromatography step followed by an ion exchange column chromatography step. The purification in addition to two steps can be performed by a combination of these different chromatographies. A step of diafiltration, ultrafiltration or gel filtration can be carried out in addition. The purity of a product after such purification can reach 99% purified product.

The proteins according to the invention can also be produced in the milk of non-human transgenic animals, such as a goat, a rabbit, the sheep, the cow or a pig. In this case, the secretion of proteins by the mammary glands, allowing its secretion in the milk of the transgenic mammal, involves the control of the expression of the proteins according to the invention in a tissue-dependent manner. Such control methods are well known to those skilled in the art. The control of the expression is carried out by means of sequences allowing the expression of the protein towards a particular tissue of the animal. These include the promoter sequences WAP, beta-casein, beta-lactoglobulin and signal peptide sequences. The process for extracting proteins of interest from the milk of transgenic animals is described in patent EP 0 264 166.

Another aspect of the invention also relates to the use of a recombinant protein according to the invention as a medicament.

The invention also relates to the use of a recombinant protein according to the invention for the manufacture of a medicament for the treatment of a disease involving an undesirable or inappropriate complement activity, in particular for the treatment of diseases due to inflammation uncontrolled or an uncontrolled C3b deposit. The invention also relates to a method of treating a disease involving an undesirable or inappropriate complement activity, comprising administering a recombinant protein according to the invention to a patient in need of such treatment. The term "treatment" includes both curative and prophylactic treatment of the disease. A curative treatment is defined as a treatment resulting in a cure or treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes the symptoms of an illness or the suffering it causes. Prophylactic treatment includes both treatment leading to disease prevention and treatment that reduces and / or delays the incidence or risk of disease. The invention aims in particular at the treatment of a disease selected from the group consisting of age-related macular degeneration (AMD), periodontitis, lupus erythematosus, lupus nephritis, dermatomyositis, myasthenia gravis, glomerulonephritis membranoproliferative (GNMP), psoriasis, multiple sclerosis and lesions of renal ischemia / reperfusion. The present invention is illustrated by the following figures and examples. These figures and examples are in no way intended to limit the scope of the present invention.

figures:

FIG. 1 is a diagram representing the construction strategy of the N-terminal fragments of the recombinant proteins according to the invention.

FIG. 2 is a diagram showing the construction strategy of the C-terminal fragments of the recombinant proteins according to the invention.

Figures 3A, 3B and 3C are graphs showing the acceleration activity of C3 convertase dissociation for H-factor fragments having the highest productivity.

Examples: Example 1: Construction of the Factor H N-ter and C-ter Fragments in the pCEP4 Plasmid

The purpose is to subclone in different forms the N-terminal and C-terminal fragments of the factor H variant Y402 in an optimized version in the pCEP4 expression vector. The fragments will both be completed 5 'of a NotI site, the kozak sequence and a signal peptide, and at 3' of a His-TAG followed by the BamHI site, the difference will be made by a NheI site. located at 3 'for the N-ter fragments and at 5' for the C-ter fragments. Explanatory diagrams will be described in the protocol below. 1 / Vector construction pCEP4-N-ter

1 / Construction of N-ter fragments

The N-ter fragments are constructed by PCR from the pCDNA2001neo-MD3Y vector. This vector corresponds to the vector pCDNA2001neo containing the nucleic acid represented by the sequence SEQ ID NO: 90 which is an optimized sequence encoding the Y402 variant of factor H comprising an artificial signal peptide PS-MB7. The construction strategy is shown in Figure 1.

The primers used are the following:

primer meaning: Pl-NT-FCTH 5 ^* -CTCTAGCGGCCGCGCGCCACC-3 ^* (SEQ ID NO: 91)

(nucleotides 6 to 13 of this sequence define a NotI restriction site)

antisense primers (or P2-NT-X primers):

Each of these primers contains, in this order of 5 'to 3': a BamHI site, 2 stop codons, a hexahistidine label, a linker (GS), a NheI site and a factor-specific sequence. These elements are represented on Figure 1.

P2-NT-4 (SEQ ID NO: 92)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCAGATTTTTCCTCGCAGGAAGGCAG 75pb

P2-NT-7 (SEQ ID NO: 93)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCAGTTTT CACGCGGATGCACCTTG 74pb

P2-NT-8 (SEQ ID NO: 94)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCAGACT TGATGCAGGTAGGCTGG 73pb

P2-NT-9 (SEQ ID NO: 95)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCTTCCC GCTCATAGCAGATGGGCAG 75pb

P2-NT-10 (SEQ ID NO: 96)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCGCTCT GCACCTGCTCCTTGCAAATAG 77pb

P2-NT-11 (SEQ ID NO: 97)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCGGTGG ATTCCTCGACGATGCAC 73pb

P2-NT-12 (SEQ ID NO: 98)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCTTTCTT CAGTTTGTCAATGGCGAC 75pb

P2-NT-13 (SEQ ID NO: 99)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGCGCCCAGCT GGATCTGTGCCATAGAGCAG 76pb

This series of P2-NT-X primer (X, variable) is obtained by PCR assembly. • PCR assembly between the P2NT primer and the P2-X primers (X, variable) below:

It is carried out by means of the following primers:

1- P2NT (SEQ ID NO: 100)

CTCTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCTGCCGCTAGC

2- P2-4 (SEQ ID NO: 101)

CTGCCTTCCTGCGAGGAAAAATCTGGCGCTAGCGGCAGCCAC

2-P2-7 (SEQ ID NO: 102)

CAAGGTGCATCCGCGTGAAAACTGGCGCTAGCGGCAGCCAC

2-P2-8 (SEQ ID NO: 103)

CCAGCCTACCTGCATCAAGTCTGGCGCTAGCGGCAGCCAC

2-P2-9 (SEQ ID NO: 104)

CTGCCCATCTGCTATGAGCGGGAAGGCGCTAGCGGCAGCCAC

2-P2-10 (SEQ ID NO: 105)

TATTTGCAAGGAGCAGGTGCAGAGCGGCGCTAGCGGCAGCC AC

2-P2-11 (SEQ ID NO: 106)

GTGCATCGTCGAGGAATCCACCGGCGCTAGCGGCAGCCAC

2-P2-12 (SEQ ID NO: 107)

GTCGCCATTGACAAACTGAAGAAAGGCGCTAGCGGCAGCCAC

2-P2-13 (SEQ ID NO: 108)

CTGCTCTATGGCACAGATCCAGCTGGGCGCTAGCGGCAGCCAC

Amplification of fragments of interest

Pl-NT-FCTH P2-NT-13 Nter 1 -13 pCDNA2001neo-MD3Y 2473

21 Vector construction

• Notl / BamHI Digestion of Inserts:

• Notl / BamHI digestion of the vector pCEP4:

→ Obtaining 2 fragments 24 and 10162bp

Nucleospin extract II (Clontech)

• Ligation and transformation of bacteria TOP 10

• Screening by PCR: CMVl / MD2-lRev → Obtaining a 594pb amplicon

CMV1 primers - SEQ ID NO: 109: 5 * -CCATTGACGTCAATGGGAGTTTG-3 * MD2-lrev - SEQ ID NO: 1 10: 5'-tgtcacactcgcggtagttg-3 '

3 / Sequencing

CMV1 and SV40-3 'UTR primers are used for sequencing all vectors. Only the pCEP4-lNTer1, pCEP4-lNTer1 2 and pCEP4-INTer1 3 vectors have additional sequencing with the MD2-3 primer.

Primer SV40-3 'UTR - SEQ ID NO: 1 1 1: 5' -TTCACTGCATTCTAGTTGTGGT-3 'II / Vector Construction pCEP4-C-ter

1 / Construction of C-ter fragments The C-ter fragments are constructed by PCR from the pCDNA2001neo-MD3Y vector. This vector corresponds to the vector pCDNA2001neo containing the nucleic acid represented by the sequence SEQ ID NO: 90. The construction strategy is shown in Figure 2.

The primers used are the following:

sense primers (or Pl-CT-X primers):

Each of these primers contains, in this order from 5 'to 3': a NotI site, a Kozack (SK) sequence, a signal peptide (PS), a NheI site and a factor H specific sequence. These elements are represented in Figure 2.

P1-CT-19 (SEQ ID NO: 112)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTGGACCCCCTCCACCCATC

P1-CT-16 (SEQ ID NO: 113)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTAAGAGTCCTCCAGAGATTTCACA

Pl-CT-15 (SEQ ID NO: 114)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTAGCCAGCCCCCTCAGATC

P1_CT-14 (SEQ ID NO: 115)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGCCCACCACCTCCACAGATTC

Pl-CT-13 (SEQ ID NO: 116)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGCAAGTCCTCTAATCTGATCATTC

P1-CT-12 (SEQ ID NO: 117)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTGGCGATATTCCAGAACTGG

Pl-CT-11 (SEQ ID NO: 118)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTGGACCACCTCCAGAACTGC

P1-CT-10 (SEQ ID NO: 119)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTGAGCTGCCAAAAATTGATG

Pl-CT-9 (SEQ ID NO: 120) CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTGACATTCCAGTGTTTATGAACG

Pl-CT-8 (SEQ ID NO: 121)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTTCTAAGAGCAGCATCGACATTG

antisense primer (P2-CT-FCTH)

This primer contains, in the 5 'to 3' orientation: a BamHI site, 2 stop codons, a hexahistidine tag, a linker (GGSG) and a specific H factor sequence.

P2-CT-FCTH (SEQ ID NO: 122)

GAGTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCCGCTTCCGCCTCTCTTAG CACAAGTAGGGTATTCCAG 74pb

The series of Pl-CT-X primer (X, variable) and the P2-CT-FCTH primer are obtained by assembly PCR.

• PCR assembly to obtain primers:

primers used to obtain the P2-CT-FCTH primer

l-P2-CT-FCTH-for (SEQ ID NO: 123)

GAGTAGGATCCTTATCAATGGTGGTGATGGTGGTGGCCGCTTCCG

2-P2-CT-FCTH-rev (SEQ ID NO: 124)

CTGGAATACCCTACTTGTGCTAAGAGAGGCGGAAGCGGCCACCAC

primers used to obtain Pl-CT-X primers

PCR1: 1-P 1 -CT-FCTH 1 (SEQ ID NO: 125)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTG

1-P1-CT-FCTH2 (SEQ ID NO: 126)

CGTTAGCAGAAGTGATAGACAGCAGCAGCAGAAAAATCCAAGACCATCGCATG

PCR1 Fragment: P1-CT-FCTH-PCR1 (SEQ ID NO: 127)

CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACG 73PB primer primer Dimer size sense anti-sense Dimer creates (Pb)

1-PL-CT-FCTH1 1 -P1-CT-FCTH2 Pl -CT-FCTH-Pcrl 73

PCR2:

2-P1-CT-19 (SEQ ID NO: 128)

GATGGGTGGAGGGGGTCCACAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P 1 -CT-16 (SEQ ID NO: 129)

TGTGAAATCTCTGGAGGACTCTTACAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P1-CT-15 (SEQ ID NO: 130)

GATCTGAGGGGGCTGGCTACAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P1-CT-14 (SEQ ID NO: 131)

GAATCTGTGGAGGTGGTGGGC AGCCGCTAGC AGCGTTAGC AGAAGTGA

2-P1-CT-13 (SEQ ID NO: 132)

GAATGATCAGATTAGAGGACTTGCAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P1-CT-12 (SEQ ID NO: 133)

CCAGTTCTGGAATATCGCCACAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P 1 -CT-11 (SEQ ID NO: 134)

GCAGTTCTGGAGGTGGTCCACAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P1-CT-10 (SEQ ID NO: 135)

CATCAATTTTTGGCAGCTCACAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P1-CT-9 (SEQ ID NO: 136)

CGTTCATAAACACTGGAATGTCACAGCCGCTAGCAGCGTTAGCAGAAGTGA

2-P1-CT-8 (SEQ ID NO: 137)

CAATGTCGATGCTGCTCTTAGAACAGCCGCTAGCAGCGTTAGCAGAAGTGA

Pl-CT-FCTH-PcrI 2-P1-CT-9 Pl-CT-9 1 10

Pl-CT-FCTH-PcrI 2-P1-CT-8 Pl-CT-8 1 10

• Amplification of fragments of interest:

21 Vector construction

• Notl / BamHI Digestion of Inserts:

Notl / BamHI digestion of the pCEP4 vector:

→ Obtaining 2 fragments 24 and 10162bp Nucleospin extract II

• Ligation and transformation of bacteria TOP 10

• Screening by PCR: CMV1 / P2-MB7 → Obtaining a 259pb amplicon

Seq P2-MB7 - SEQ ID NO: 138: 5'-

TGGTGATGCTCAGCAGCAGCAGGAAGATCCAGCTCCATCG-3 '

3 / Sequencing CMV1 and SV40-3'UTR primers are used for the sequencing of all the vectors. Only the pCEP4-9Cter20 and pCEP4-8Cter20 vectors will have additional sequencing with the MD2-5 primer.

Seq MD2-5 - SEQ ID NO: 139: 5'-GGATTCACACCGTGTGCATTAATG-3 'Example 2: Construction of the vectors Factor H combining the domains N-ter and C-ter

From the 8 pCEP4-lNTX vectors and the pCEP4-XCT20 vectors, we construct 59 vectors combining the N-ter and C-ter fragments, containing at least the N-terminal SCR1-4 domains and the SCR19-domains. C-terminals to which are added a variable number of SCRs domains in the central part of the molecule.

First, we introduce the 1NTX fragments, present in the plasmid pCEP4 in the pCDNA2001neo vector (by NotI / BamHI digestion).

In a second step, we introduce into the pCDNA2001neo-1NTX vectors the XCT20 fragments (by NheI / BamHI digestion).

We thus obtain 59 plasmid constructs which correspond to the 59 combinations 1NTX-XCT20 of the fragments FH. These sequences are present in the pCDNA2001neo vector which allows stable expression of these molecules in the PERC6 cell line. To facilitate the screening and production work, the FH 1NTX-XCT20 fragments are extracted from the pCDNA2001neo vector by NotI / BamHI digestion and cloned into the pCEP4 vector which allows transient expression in HEK293F cells.

1 / Construction of pCDNA2001neo-N-Ter vectors from pCEP4-N-ter vectors Digestion of pCEP4-lNT X vectors by NotI / BamHI:

Gel purification of the fragment of interest followed by a Nucleospin extract II.

Digestion of the pCDNA200 lneo vector by Not I / BamHI.

Nucleospin extract II.

· Ligation and transformation TOP 10.

• PCR screening: CMV1 / MD2-1REV Obtaining a 706 bp fragment.

• Control by Notl / BamHI digestion: checking the presence of the insert.

II / Construction of pCDNA2001neo-lNTX / XCT20 vectors for production in the PERC6 cell line:

8CT20 13CT20

19CT20 12CT20

16CT20 19CT20

15CT20 16CT20

14CT20 1NT12 15CT20

1NT8 13CT20 14CT20

12CT20 13CT20

11CT20 19CT20

10CT20 16CT20

1NT13

9CT20 15CT20

14CT20

• Digestion of the vectors pCEP4-XCT20 by NheI / BamHI:

pCEP4-19CT20 414 bp pCEP4-16CT20 948 bp pCEP4-15CT20 1131 bp pCEP4-14CT20 1308 bp pCEP4-13CT20 1482 bp pCEP4-12CT20 1668 bp pCEP4-HCT20 1851 bp pCEP4-10CT20 2034 bp pCEP4-9CT20 2214 bp pCEP4-8CT20 2397 bp

Digestion of the pCDNA200 lneo- 1NTX vector with Nhe I / BamHI. Nucleospin extract II.

• Ligation and transformation TOP 10.

• PCR screening: MD2-6 / 2BGHPA Obtaining a 373 bp fragment.

NotI / BamHI and NheI / BamHI control digestion.

Seq MD2-6 - SEQ ID NO: 140: 5'-AGGGAAACAAGCGCATCACCT-3 '

Seq 2BGHPA - SEQ ID NO: 141: 5 '-CAGATGGCTGGCAACTAGAA-3'

The 1NTX / XCT20 fragments are then extracted by NotI / BamHI digestion and reintroduced into the pCEP4 vector.

III / Construction of pCEP4-lNTX / XCT20 vectors for production in the HE 293 Freestyle cell line:

Digestion of the vector pCEP4 by NotI / BamHI (10162 and 24bp)

Nucleospin extract II.

• Digestion of the pCDNA200 lneo-1NTX / XCT20 vectors by NotI / BamHI (size of the fragments in the table below.)

Gel purification

Nucleospin extract II.

Size Size

Digested vector (NotI / BamHI) digested vector insert (NotI / BamHI) insert

(pb) (pb) pCDNA200 lneo-1NT4 / 19CT20 1216 pCDNA200 lneo-1NT9 / 19CT20 2134 pCDNA200 lneo-1NT4 / 16CT20 1762 pCDNA200 lneo-1NT9 / 16CT20 2668 pCDNA200 lneo-1NT4 / 15CT20 1945 pCDNA200 lneo-1NT9 / 15CT20 2851 pCDNA200 lneo 1NT4 / 14CT20 2122 pCDNA200 lneo-1NT9 / 14CT20 3028 pCDNA200 lneo-1NT4 / 13CT20 2296 pCDNA200 lneo-1NT9 / 13CT20 3202 pCDNA200 lneo-1NT4 / 12CT20 2482 pCDNA200 lneo-1NT9 / 12CT20 3388 pCDNA2001neo-1NT4 / 1CT20 2665 pCDNA2001neo-1NT9 / l 1CT20 3571 pCDNA200 lneo-1NT4 / 10CT20 2848 pCDNA200 lneo-1NT9 / 10CT20 3754 pCDNA2001neo-lNT4 / 9CT20 3028 pCDNA200 lneo-INT 10 / 19CT20 2317 pCDNA2001neo-lNT4 / 8CT20 3211 pCDNA200 lneo-INT 10 / 16CT20 2851 pCDNA200 lneo-1NT7 / 19CT20 1771 pCDNA200 lneo- INT 10 / 15CT20 3034 pCDNA200 lneo-1NT7 / 16CT20 2305 pCDNA200 lneo-INT 10 / 14CT20 3211 pCDNA200 lneo-1NT7 / 15CT20 2488 pCDNA200 lneo-INT 10 / 13CT20 3385 pCDNA200 lneo-1NT7 / 14CT20 2665 pCDNA200 lneo - INT 10 / 12CT20 3571 pCDNA200 lneo-1NT7 / 13CT20 2839 pCDNA200 lneo-INT 10 / 11CT20 3754 pCDNA200 lneo-1NT7 / 12 CT20 3025 pCDNA200 lneo- INT 11 / 19CT20 2500 pCDNA2001neo-lNT7 / l 1CT20 3208 pCDNA200 lneo- INT 11 / 16CT20 3034 pCDNA200 lneo- 1NT7 / 10CT20 3391 pCDNA200 lneo- INT 11 / 15CT20 3217 pCDNA2001neo-lNT7 / 9CT20 3571 pCDNA200 lneo-INT 11 / 14CT20 3394 pCDNA2001neo-lNT7 / 8CT20 3754 pCDNA200 lneo- INT 11 / 13CT20 3568 pCDNA200 lneo- 1NT8 / 19CT20 1954 pCDNA200 lneo- INT 11 / 12CT20 3754 pCDNA200 lneo- 1NT8 / 16CT20 2488 pCDNA200 lneo-INT 12 / 19CT20 2686 pCDNA200 lneo - 1NT8 / 15CT20 2671 pCDNA200 lneo- INT 12 / 16CT20 3220 pCDNA200 lneo- 1NT8 / 14CT20 2848 pCDNA2001neo-lNT12 / 15CT20 3403 pCDNA200 lneo-1NT8 / 13CT20 3022 pCDNA200 lneo- INT 12 / 14CT20 3580 pCDNA200 lneo-1NT8 / 12CT20 3208 pCDNA2001 neo - INT 12 / 13CT20 3754 pCDNA2001neo-lNT8 / l 1CT20 3391 pCDNA200 lneo- INT 13 / 19CT20 2860 pCDNA200 lneo- 1NT8 / 10CT20 3574 pCDNA200 lneo- INT 13 / 16CT20 3394 pCDNA2001neo-lNT8 / 9CT20 3754 pCDNA200 lneo-INT 13 / 15CT20 3577 pCDNA200 lneo- INT 13 / 14CT20 3754 • Ligation and transformation TOP 10.

• PCR Screening: CMV1 / MD2-1REV Obtaining a 594 bp Fragment.

• Sequencing of the junctions by CMV 1 and S V40-3 'UTR. Example 3 Determination of the Concentration and Molecular Weight of the FH Fragments Present in the Supernatants of HEK 293F Cells After 7 Days of Batch Mode Production

3.1: Transient transfection in HEK 293F cells for transient production of recombinant FH fragments.

1 - Transient transfection:

On the day before the transient transfection, the HEK 293F cells are subcultured at a cell concentration of 7 ^E 5 vc / ml. The cell density and viability of HEK 293F cells are determined on the day of transfection. The culture volume corresponding to 30 ^E 6 cv / ml is centrifuged. The supernatant is removed and the cell pellet is taken up in 28 ml of F17 culture medium (Invitrogen), transferred to a 250 ml Erlenmeyer flask and incubated at 37 ° C.

2- Formation of the transfection agent / DNA complex in ratio 2: 1:

The transfection agent and the DNA corresponding to the vector pCEP4 containing one of the sequences of the FH fragments are prepared in OptiMEM medium (Invitrogen) as follows:

- Addition of 30μg of DNA in 1 ml of OptiMEM

- Add 60 μΐ of Fectine or 60 μΐ of PEI in 1 ml of OptiMEM.

These two preparations are incubated separately for 5 minutes at room temperature and then the solution containing the transfection agent is added gently to that containing the DNA. The mixture is incubated for 25 minutes at room temperature before being added to the 28 ml of HEK 293F cells previously prepared. The cells are then incubated at 37 ° C. with shaking at 125 rpm. 3 -Transitional production of factor H:

The cells are kept in culture for 7 days without adding or renewing culture medium. The ^7th day, the cells were centrifuged at 3000g for 15 minutes. The cell pellets are removed and the cell supernatants containing the recombinant FH fragments are filtered in 0.22 μι ρυίβ frozen at -20 ° C. 3.2: Assaying human FH fragments in the culture medium by ELISA

Antibody coating antibody: human anti-Human H mutagen (The Binding Site) diluted extemporaneously in PBS buffer pH 7.4 to obtain a concentration of between 3.5 and 5.5 μg / ml.

Saturation buffer: PBS - BSA 1% (p / vol)

Wash Buffer: PBS - Tween-20 0.1% (vol / vol)

Dilution buffer: PBS buffer - Tween-20 0.1% (vol / vol) - 0.1% BSA (w / vol)

Standard solution: HR Human H Calibrator (The Binding Site)

Samples: the samples are prediluted so as to obtain a concentration close to that of the first point of range. Then dilute ½ to ½.

Monoclonal Antibody Anti Factor H: Monoclonal Immunoglobulins from Purified Mouse Anti Human Factor H (SEROTEC)

Revealing antibody: Peroxidase-labeled mouse anti-mouse IgG goat immunoglobulins (Jackson Immuno Research Laboratories).

Revealing Solution: TMB Kit (Pierce)

Stop Solution: 4 N or 2 M sulfuric acid (Fisher Scientifïc).

OPERATIVE MODE

Sensitization of the solid phase:

In a microtiter plate, 100 μΐ per well of diluted coating antibody is dispensed. The plate is then covered with an adhesive sheet and then incubated overnight at + 4 ° C. The plate is then emptied by inversion. Saturation of the solid phase:

One distributes 120 μΐ per well of saturation solution. The plate is then covered with an adhesive sheet and then incubated for 1 hour at 20 ° C. The washings are then washed 3 times. Antigen capture:

100 μΐ per well of dilution buffer (blank), each dilution of standard and samples are dispensed. The plate is then covered with an adhesive sheet and incubated for 1 hour 30 minutes at 20 ° C. Then washed 5 times in washing buffer. Recognition of the antigen by the monoclonal antibody:

100 μl of the monoclonal antibody is distributed in each well. The plate is covered with an adhesive sheet and then incubated incubated for 1 hour 30 minutes at 20 ° C. Then washed 5 times in washing buffer.

HRP Conjugate Marking (HorseRadish Peroxidase in English, or Horseradish Peroxidase):

100 μl of the revealing antibody are distributed, the plate is covered with an adhesive sheet, then incubated for 1 hour at 20 ° C. The wells are washed 5 times in washing buffer.

Enzymatic reaction:

100 μΐ per well of TMB containing the substrate is dispensed at a regular time interval and away from any intense light. Then incubate at room temperature between 5 - 15 minutes. This time must be identical for all dosing points.

Stopping the reaction:

100 μl of H ₂ S0 ₄ 4N are dispensed per well at a regular time interval. The results are reported in the following table.

1NT7-12CT20 42 111244.2

1NT7-11CT20 154 118127.1

1NT7- 10CT20 157 124969.1

1NT7- 9CT20 161 131845.6

1NT7- 8CT20 224 138405.9

1NT8- 19CT20 7 70758.5

1NT8- 16CT20 47 90603.9

1NT8-15CT20 124 97299.2

1NT8- 14 CT20 339 103969.8

1NT8- 13 CT20 48 110808.6

1NT8-12 CT20 13 117804.6

1NT8- 11 CT20 115 124687.4

1NT8- 10 CT20 128 131529.4

1NT8- 9 CT20 80 138405.9

1NT9-19 CT20 5 77635.1

1NT9-16 CT20 8 97480.4

1NT9-15 CT20 43 104175.7

1NT9-14 CT20 91 110872.3

1NT9-13 CT20 25 117711.2

1NT9-12 CT20 25 124655.0

1NT9-11 CT20 45 131537.9

1NT9-10 CT20 156 138405.9

1NT10- 19CT20 5 84451.0

1NT10- 15CT20 13 110991.7

1NT10- 14CT20 21 117688.3

1NT10- 11CT20 128 138379.9

1NT11-19CT209 91333.8

1NT11- 16CT20 22 111179.2

1NT11- 15CT20 16 117874.5

1NT11- 14CT20 36 124571.1

1NT13- 14CT20 13 138379.9

Example 4: Characterization by SDS-PAGE of the recombinant FH fragments present in the culture supernatant (FIGS. 6A, 6B and 7).

From the value of the concentration of the recombinant FH fragments determined by ELISA, a volume corresponding to 1 g of recombinant factor H is diluted to ½ in 2x Laemmli buffer, heated at 95 ° C. for 5 minutes and then deposited on a linear gel. 10% polyacrylamide. After migration, the proteins contained in the gel are stained with Coomassie blue. Analysis of the polyacrylamide gels shows that the recombinant FH fragments migrate according to the apparent molecular masses expected.

EXAMPLE 5 Determination of the C3 convertase dissociation accelerating activity of the recombinant FH fragments present in the culture supernatant and having the highest productivity:

From the recombinant factor H concentration determined by ELISA, the recombinant FH fragments are diluted to a final concentration of 20 μg / ml. From this tube a range is achieved by the following successive dilutions: 1/2; 1/10; 1/4; 1/4; 1/4; 1/4; 1/40. This decreasing concentration range of FH fragments is added to the C3 convertase complex formed in the wells of a 96-well plate which is then incubated 32-34 minutes at + 34 ° C. After several washes, the factor B remaining complexed with the immobilized C3 molecule at the bottom of the well is then assayed by an immunoenzymatic reaction of the ELISA type. Absorbance values obtained as a function of the concentration of added FH fragments are then processed in a nonlinear modeled system (variable slope sigmoid) to determine the IC50 value of each sample. The calculation equation of this model is as follows:

Y: Bottom + (Top -bottom) / (l + 10 ^A ((log IC50-x) * hillslope)

X is the logarithm of the concentration (g / ml).

Y is the answer (OO).

It starts at the baseline and goes to the top in a sigmoidal form. Hillslope is the slope.

The activity determined in the cell supernatant is shown in Figures 3A, 3B and 3C for the fragments having the highest productivity.

Example 6 Determination of the Acceleration Activity of the Dissociation of C3 Convertase of Purified Recombinant FH Fragments

The FH fragments were purified via the hexahistidine tag which was added to the c-terminus of the proteins. The purification was carried out in an affinity chromatography step using HisTALON (Clontech) columns which contain a resin having high affinity and specificity for polyshistidins. The purification was carried out according to the supplier's recommendations (HisTALON Gravity Column Purification Kit User Manual). After purification the samples are dialyzed against PBS buffer and stored at

4 ° C.

The concentration of the purified recombinant FH fragments is determined by measuring the OD at 280 nm. The purified recombinant FH fragments are diluted to a final concentration of 150 nM and then from this tube a range is achieved by the following successive dilutions: 1/2; 1/10; 1/4; 1/4; 1/4; 1/4; 1/40. This decreasing concentration range of FH fragments is added to the C3 convertase complex formed in the wells of a 96-well plate which is then incubated 32-34 minutes at + 34 ° C. After several washes, the factor B remaining complexed with the immobilized C3 molecule at the bottom of the well is then assayed by an ELISA-type immunoenzymatic reaction, using TMB (3,3 ', 5,5'-tetramethylbenzidine) or OPD (ortho-phenylenediamine) as reaction substrate. Absorbance values obtained as a function of the concentration of added FH fragments are then processed in a nonlinear modeled system (variable slope sigmoid) to determine the IC50 value of each sample. The calculation equation of this model is as follows:

Y: Bottom + (Top -bottom) / (l + 10 ^A ((log IC50-x) * hillslope)

X is the logarithm of the concentration (g / ml).

Y is the answer (OO).

The IC ₅ o values obtained are shown in the table below.

pCEP4- 1NT4-19CT20 0.0916 73% pCEP4- 1NT7- 8CT20 0.5419 72% pCEP 4-lNT9-l l CT20 0.1716 69% pCEP 4-lNT9-10CT20 0.5517 64% pCEP 4-lNT9-14 CT20 0.5698 64% pCEP4- 1NT8-14CT20 0.6454 61% pCEP4- 1NT7-10CT20 0.6015 59% pCEP4- 1NT10- 11CT20 0.6118 58% pCEP4- 1NT8- 15CT20 0.6279 58% pCEP4- 1NT4- 15CT20 0.2061 58% pCEP 4-lNT9-13 CT20 0.1663 56% pCEP4- 1NT7-15CT20 0.6393 56% pCEP 4-lNT9-19 CT20 0.2166 49% pCEP4- 1NT13- 14CT20 0.7765 45% pCEP4- 1NT4- 8CT20 0.2276 42% pCEP4- 1NT4- 10CT20 0.2439 38% pCEP 4-INT9-12 CT20 0.3070 30% pCEP4- 1NT8- 13 CT20 0.4022 30% pCEP4- 1NT8- 9 CT20 0 , 4886 27% pCEP 4-lNT9-16 CT20 0.4177 25% pCEP4- 1NT8- 16CT20 0.4264 25% pCEP4- 1NT4- 9CT20 0.5360 18% pCEP4- 1NT4- 11CT20 0.5458 17% pCEP4- 1NT4- 14CT20 0.6179 15% pCEP4- 1NT7- 16CT20 0.4418 15% pCEP4- 1NT10- 14CT20 0.6714 14% pCEP4- 1NT7- 19CT20 1.0900 12% pCEP4- INTl l- 15CT20 0.7395 10% pCEP4- INTl l- 16CT20 1.2120 8% pCEP4- 1NT7- 13CT20 1.9260 7% pCEP4- 1NT7- 12CT20 2.0350 7% pCEP4- 1NT10- 15CT20 1.1680 6% pCEP4- INT 1 pCp4-INT1 1 -19CT20 1.9900 5% pCEP4-INT9-15 CT20 2.1880 5% pCEP4-1NT8-12 CT20 1.8700 4% pCEP4-1NT10-19CT20 6, 0520 1% pCEP4- 1NT8- 19CT20 493.0000 0% The activity of an entire recombinant H factor produced in PerC6 or HEK cells and purified was used as an activity control. The C3 activity of the whole H factor produced in PerC6 cells is equivalent to that of the whole H factor produced in HEK cells.

Example 7: Determination of the protection activity of lysis of red blood cells of sheep by modified Sanchez-C orrai test.

This test makes it possible to measure the functional activity of the C-terminal portion of the purified human recombinant H factor during the development of therapeutic batches by evaluating its ability to protect sheep red blood cells from lysis induced by a depleted or factor-deficient serum. H functional. This test is adapted from the "Sanchez-Corral" method to measure the anti-hemolytic activity of factor H present in the plasma of patients with hemolytic uremic syndrome. (P. Sanchez-Corral, C. Gonzalez-Rubio, S. Rodriguez of Cordoba and M. Lopez-Trascasa, Molecular Immunology 41 (2004) 81-84).

In this context, a mixture of H factor depleted serum and a human plasma pool is produced in equal proportion to create the conditions for a specific lysis. The addition of purified human recombinant H factor ensures the protection of sheep red blood cells (lack of cell lysis) against complement-induced lysis.

This test was used here to determine the protective activity of red cell lysis of factor H fragments according to the invention.

Determination of the percentage of lysis:

To 40 μl of a suspension of sheep red blood cells (1 × 10 ⁸ red blood cells / ml) are successively added 20 μl of the reaction buffer (10 mM Hepes, 144 mM NaCl, 7 mM MgCl 2, 10 mM EGTA, pH 7.2). variable volume of FH fragment or whole FH (0-30μ1) corresponding to concentrations of 0 to 44.74pM, then 9 μΐ of a pool of human plasma followed by 9 μΐ of human plasma depleted in FH. The reaction volume is completed with PBS buffer to obtain a final volume of 1 ΙΟμΙ. After incubation for 30 minutes at 37 ° C., 400 μl of HBS-EDTA buffer (10 mM Hepes, 144 mM NaCl, 2 mM EDTA, pH 7.2) were used to stop the reaction. After centrifugation for 5 min at 1730 g, 200 μl of supernatant are removed and deposited in a microtiter plate in order to measure the absorbance at 414 nm.

The percentage of lysis is determined according to the formula:

The "100% lysis" control corresponds to the maximum lysis of sheep red blood cells observed in the presence of water.

The blank control comprises the + 50mM EDTA reaction buffer and corresponds to the spontaneous lysis of sheep red blood cells.

Without CFH, spontaneous lysis of red blood cells is about 30%.

In the following tables, the analysis is made by normalizing the value obtained with the plasma H factor (FH LP03 0 pm) to 100%. The negative values obtained are normalized to the value of 0%.

44.74 2.5 3.7 1.1 0

The tests show similar results for rFH samples PERC6 (14-

HFACEX-1446-042) and rFH HEK (12-HFACEX-1446-072) and consistent with plasma FH (control LP03): dose-effect with total protection of hemolysis at the highest concentration of factor H.

The various fragments of factor H according to the invention are very active with an inhibition of specific lysis greater than the entire rFH from the lowest concentration tested. For two fragments, 1NT9-16CT20 and 1NT7-16CT20, a profile closer to the plasma H factor LP03 is very interestingly observed.

Claims

A recombinant protein having a factor H activity, comprising, from the N-terminus to the C-terminus, a first amino acid sequence comprising at least the domains SCR1 to SCR4 of the factor H, and a second amino acid sequence comprising at least the SCR19 and SCR20 domains of the factor H, said recombinant protein not being a natural factor H, and provided that if the first sequence consists of the domains SCR1 to SCR4 and the second sequence is constituted domains SCR19 and SCR20, the linker is a synthetic linker.

The recombinant protein of claim 1, first and / or second amino acid sequence further comprising one or more other rearranged H factor SCR domains.

3. Recombinant protein according to claim 1 or 2, the first and the second amino acid sequence being linked together by a non-natural linker, in particular a G-A-S-G linker.

A recombinant protein according to any one of the preceding claims, wherein the first or second amino acid sequence comprises at least the SCR12, SCR13 and SCR14 domains of factor H, or the first and second amino acid sequences do not include any of the domains SCR12, SCR13 and SCR14.

A recombinant protein according to any one of the preceding claims, the first sequence comprising the domains SCR1 to SCR4 of the factor H, and the second sequence is selected from the group consisting of:

- domains SCR16 to SCR20,

- domains SCR15 to SCR20;

- domains SCR10 to SCR20; and

domains SCR8 to SCR20 of the factor H.

A recombinant protein according to any one of the preceding claims, the first sequence comprising domains SCR1 to SCR7 of factor H, and the second sequence comprising domains SCR1 to SCR20 of factor H, said second sequence preferably being selected from group consisting of:

- domains SCRl 5 to SCR20

domains SCR1 4 to SCR20;

domains SCR1 1 to SCR20;

- domains SCR10 to SCR20;

- domains SCR9 to SCR20; and

domains SCR8 to SCR20 of the factor H.

7. Recombinant protein according to any one of the preceding claims, the first sequence comprising domains SCR1 to SCR8 of factor H, and the second sequence comprising domains SCR1 6 to SCR20 of factor H, said second sequence preferably comprising domains SCR10. at SCR20 of factor H.

A recombinant protein according to any one of the preceding claims, the first sequence comprises domains SCR1 to SCR4 of factor H and the second sequence comprises domains SCR1 to SCR20 of factor H, a third sequence comprising domain SCR7 of factor H being between the first and the second sequence.

A recombinant protein according to any one of the preceding claims, the first amino acid sequence comprising an N-terminal signal peptide, especially the natural signal peptide of factor H (SEQ ID NO: 24), the signal peptide of a protein different from the factor H, or the signal peptide PS-MB7 represented in the sequence SEQ ID NO: 25.

10. The recombinant protein according to any one of the preceding claims, of a sequence selected from the sequences represented in SEQ ID NO: 26 to 84 and 159.

11. A nucleic acid construct encoding a recombinant protein according to any one of claims 1 to 10.

The nucleic acid construct of claim 11, comprising a unique restriction site between the two nucleic acid sequences encoding the first and second amino acid sequences of the recombinant protein, including a Nhe I site present in the encoding a GASG linker.

A vector comprising a nucleic acid construct according to claim 11 or 12.

A host cell comprising the nucleic acid construct of claim 11 or 12, or the vector of claim 13.

A transgenic non-human animal comprising the nucleic acid construct of claim 11 or 12, or the vector of claim 13.