EP3397266A1

EP3397266A1 - Factor h fragment for use as an anti-angiogenic agent

Info

Publication number: EP3397266A1
Application number: EP16829428.8A
Authority: EP
Inventors: Virginie DINET; Frédéric MASCARELLI; Toufik ABACHE; Sylvie Jorieux
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris 5 Rene Descartes; Universite Paris Diderot Paris 7; LFB SA; Sorbonne Universite
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris 5 Rene Descartes; Universite Paris Diderot Paris 7; LFB SA; Sorbonne Universite
Priority date: 2015-12-30
Filing date: 2016-12-30
Publication date: 2018-11-07
Also published as: US20190388506A1; FR3046355A1; WO2017115059A1

Abstract

The invention relates to a complement factor H fragment for use in the therapy and/or prophylaxis of a disease involving neovascularization.

Description

Fragment of factor H for use as an antiangiogenic agent

The present invention relates to a complement factor F (FH) fragment for its use in the treatment and / or prevention of a pathology involving neovascularization.

Angiogenesis is a complex physiological mechanism that plays a major role in the genesis of neovascularization involved in various pathologies including the growth of solid tumors, atherosclerosis, rheumatoid arthritis, psoriasis and intraocular neovascular diseases such as retinopathies. proliferative or age-related macular degeneration (AMD) exudative.

Inhibitors of vascular endothelial growth factor (VEGF) are used in the treatment of various cancers and ocular conditions. Currently available as VEGF inhibitors are pegaptanib, bevacizumab, ranibizumab, or aflibercept. Although these molecules represent a breakthrough in the treatment of exudative AMD, it turns out that the recovery of vision obtained through these treatments is only transient, retinal edema secondary to neovascularization recurrence and retinal degeneration occurs. continues. In addition, 20-30% of patients respond poorly to these treatments (Rasmussen and Sander, 2014) and show residual exudative signs.

On the other hand, if the excess of VEGF has a promoter effect of the neovascularization and the associated edema, in counterpart, a total inhibition can have a detrimental effect on the neural retina, the choroid and the cells of the epithelium pigmentary retina (EPR), and especially its tight junctions, essential to maintain the integrity of the EPR. Indeed, VEGF is considered a trophic factor for the survival of EPR cells (Byeon et al., 2010), maintenance of choriocapillaries (Saint-Geniez et al., 2009) and visual functions (Saint-Geniez et al., 2009). Geniez et al., 2008). Thus, treatment by agents Anti-VEGF may result in long-term progressive atrophy of the neural retina, choroid, and EPR in patients with exudative AMD.

Moreover, an increase in the rate of progression of atrophy of the EPR has been reported in patients with exudative AMD and treated with anti-VEGF, which underlines the insufficient effectiveness of this treatment to prevent visual consequences. long-term AMD (Rosenfeld et al., 2011, Lois et al., 2013, Kumar et al., 2013, Grunwald et al., 2014, Young et al., 2014, Bhisitkul et al., 2015, Channa et al. al., 2015, Schutze et al., 2015).

In addition, it has been shown that polymorphisms located on the gene of the

FH result in resistance in some patients treated with anti-VEGF antibodies (Kloeckener-Gruissem et al., 2011; Brantley et al., 2007)

There is therefore a need to develop new therapies combining efficacy and acceptability for the treatment of pathologies involving neovascularization, and in particular ocular pathologies. Surprisingly, the inventors have identified, for the first time, a new function of the FH. After long studies, they have demonstrated the ability of FH fragments to exert anti-angiogenic activity. Such activity is particularly promising for the treatment of pathologies involving neovascularization. Thus, in a first aspect, the invention relates to a fragment of FH for its use in the treatment and / or prevention of a pathology involving neovascularization, said fragment of FH comprising the amino acid sequence represented in SEQ ID N 2.

Indeed, the inventors have demonstrated that the fragments of the FH comprising at least the SCR7 domain, represented in SEQ ID No. 2, inhibits neovascularization, in particular choroidal neovascularization. Also, H factor fragments appear as a new relevant and effective solution for the treatment of pathologies, in particular ocular pathologies, related to excessive vascularization or choroidal neovascularization. The invention therefore proposes a new therapeutic strategy effective for the treatment of patients suffering from ocular pathologies involving excessive vascularization or choroidal neovascularization.

By "Factor H" or "FH" is meant a glycoprotein involved in the regulation of the alternate pathway of complement. More specifically, FH plays a role in the regulation of the alternative complement pathway by interacting primarily with the C3b molecule. FH, encoded by the FH gene, is predominantly synthesized in the liver but can also be expressed locally by different cell types including EPR cells, endothelial cells, and others. FH has repetitive sequences of about 60 amino acids called "SCR domains" (short consensus repeat). The region of the FH involved in the fluid phase regulation of the complement activity of the alternate channel is located at the level of the SCR1-4 domains. FH has C3b binding sites (SCR1-4, SCR6-8, SCR12-14 and SCR19-20) and glycosylated glycans (GAGs) (SCR6-8, and SCR19-20). Thus, FH can interact with extracellular matrices. The domain SCR19-20 is also a link site to C3b and more specifically to C3d. FH also has C-reactive or CRP binding sites (SCR6-8 and SCR 16-20), which is an acute phase protein synthesized primarily by the liver and plays an important role in inflammatory and serves as a biological marker for these. Finally, the FH has a Zinc binding site (SCR6-8), important for the oligomerization of CFH. The FH and the Factor I (FI) regulate the alternate path of the complement by the control of the amplification loop of the alternate channel. FH binds to C3b competitively with factor B (FB), resulting in accelerated dissociation of alternating C3 (C3bBb) or C5 (C3bBbC3b) convertase, which loses its activity. In addition, C3b-linked FH acts as a co-factor for IF, allowing it to cleave C3b into C3bi, a molecule unable to bind to FB to form C3bBb. Deregulation or inactivation of the alternate pathway controlled by FH may result in uncontrolled activation of C3, resulting in the assembly of the terminal components of the complement membrane attack complex or MAC. This complex, which is composed of the C5b9 complement proteins, remains bound and forms a transmembrane pore, a cytotoxic component of the complement system, which causes lysis of the target cells.

The prior art describes that FH can be used to treat certain retinal degenerations because of its anti-inflammatory or antioxidant activity. Indeed, the document WO2006 / 088950 describes a polymorphism in the FH gene generating the Y402H variant which is associated with the increased risk of developing AMD (Edwards et al., 2005; Hageman et al., 2005; Haines et al. et al., 2005. Klein et al., 2005, Despriet et al., 2006). In addition, document WO2011 / 113641 describes the use of FH in the treatment and / or prevention of several pathologies, including ocular pathologies. This application describes a HF activity that relies on its ability to bind to endogenous malondialdehyde, thereby preventing the production of proinflammatory substances. Thus, the activity of the FH described in this document is an anti-inflammatory activity, the anti-angiogenic activity of the FH is not described. In addition, the strategy proposed by WO2011 / 113641 only aims to treat pathologies comprising an inflammatory component.

Furthermore, the document WO2013 / 140104 describes an antioxidant role of

F H. In particular, it describes that the FH protects the EPR subjected to oxidative stress, in particular through a protective effect on the tight junctions of the EPR cells against oxidative stress, more precisely against the destabilization and disorganization of the tight junctions. retinal cells subjected to oxidative stress. Here again, the anti-angiogenic activity of FH is not described.

In the article by Kim et al. (2013), it is shown that human plasma FH has an anti-angiogenic role in a rat model of laser-induced neovascularization. The present invention differs from this teaching in identifying for the first time fragments of FH, and not the entire FH, with anti-angiogenic activity.

The use of FH fragments, and not the entire FH, has several advantages. The lower molecular weight of the fragments, compared to the whole protein, allows the injection of a larger molar concentration of active molecules into the eye. Indeed, intraocular administration volumes in AMD treatments are limited (approximately 50 μΐ in humans). It is therefore advantageous to use smaller active ingredients, in order to be able to increase their numbers, formulated in a small injection volume. In addition, the use of fragments makes it possible to limit the glycosylation sites, thus allowing a limitation of the glycosylation variations during their industrial production and thus to ensure, on a large scale, a homogeneity of structure. Moreover, the inventors have shown that the fragments of the FH according to the invention not only reduce the surface of neovascularization (ie formation of new vessels) but also make it possible to reinforce the membrane integrity of the neovessels, thus limiting the diffusion of liquid or blood.

The peptide sequence of native human FH is shown in SEQ ID NO: 1. This sequence is known to those skilled in the art and is available under UniProtKB accession number - P086031 in the uniProt database.

By "FH fragment" is meant a portion of the peptide sequence of factor H. By definition, the term "fragment of factor H" excludes the entire factor H. Preferably, this fragment of FH has retained the same biological property of interest as human FH. In other words, it is a part of the peptide sequence of FH exhibiting the anti-angiogenic property of FH. By "biological property of interest of the FH" is meant the anti-angiogenic property of said FH.

By "fragment of the FH according to the invention" is meant fragments of the FH comprising at least the SCR domain 7 of human FH. The sequence of SCR7 of human FH is represented in sequence SEQ ID No. 2. It is as follows:

CYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTC MENGWSPTPRC.

This sequence is encoded by the nucleotide sequence SEQ ID No. 3.

In a preferred embodiment, the fragment according to the invention preferably comprises the SCR domain 7, fused with the N-terminal or C-terminal domains of the FH.

In another preferred embodiment, said N-terminal or C-terminal domains of the FH consist respectively of domains SCR 1 to 4 and SCR 19 to 20.

In another preferred embodiment, the fragment according to the invention preferably comprises an amino acid sequence chosen from: the sequence of the SCR7 domain of human FH, as represented in SEQ ID No. 2 and encoded by the sequence nucleotide SEQ ID NO: 3;

the sequence of SCR domains 1 to 7 of human FH, as represented in SEQ ID No. 4, and encoded by the nucleotide sequence

SEQ ID NO: 5;

the sequence of SCR domains 1 to 18 of human FH, as represented in SEQ ID No. 6, and encoded by the SEQ nucleotide sequence

ID NO: 7;

the sequence of the SCR domains 7 to 20 of human FH, as represented in SEQ ID No. 8, and encoded by the nucleotide sequence SEQ ID No. 9;

the sequence of SCR domains 7 to 18 of human FH, as represented in SEQ ID No. 10, and encoded by the nucleotide sequence SEQ ID No. 11,

the sequence of the SCR domains 6 to 8 of human FH, as represented in SEQ ID No. 12, and encoded by the nucleotide sequence SEQ ID No. 13. By "polynucleotide according to the invention" is meant a nucleic acid sequence encoding a factor H fragment according to the invention. Typically, the polynucleotide according to the invention comprises at least one coding sequence for the SCR7 domain of factor H as represented in SEQ ID No. 3.

Preferably, the polynucleotide of the invention comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO. 13.

The inventors have identified, for the first time, a novel function of FH fragments as an antiangiogenic agent and the use of such an FH fragment for the treatment of ocular pathologies involving neovascularization. In the context of the present invention, the term "pathologies involving neovascularization" encompasses all pathologies that are characterized by abnormal angiogenic activity, i.e. the formation of new blood vessels irrigating a tumor or any other tissue abnormality, including ocular. In the context of the invention, the terms neovascularization and angiogenesis are synonymous and denote an abnormal and / or excessive vascularization and / or likely to lead to a pathological situation. Such neovascularization is found in several ocular pathologies, including exudative AMD.

According to the invention, the term "treatment" or "treating" is understood to mean the action of suppressing, reducing or inhibiting the progression of the disease to which this term applies. The term "treatment" or "treating" is also understood to mean the action of suppressing, reducing, inhibiting the progression of the appearance of one or more symptoms of the condition or disease to which this term applies.

According to the invention, the term "prevention" or "prevention" means the action of avoiding, delaying or limiting the appearance of a pathology or the symptoms of a pathology in a healthy subject, or a subject known to have a predisposition to trigger said pathology. In a preferred embodiment, the pathology involving neovascularization is ocular pathology selected from AMD, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, ischemic retinopathy, retinopathy. radiation, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, venous vascular occlusion, non-tumor proliferative vasculopathies and cancer.

In another preferred embodiment, the pathology involving neovascularization is ocular pathology selected from AMD, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, and cancer.

In a preferred embodiment of the invention, said cancer is selected from the group consisting of vascularized and / or vascular intraocular tumors, proliferative tumor vasculopathies, vascular tumors of vascular or non-vascular origin such as choroidal hemangiomas, proliferations. intraretinal angiomatous, chorioretinal anastomoses or polypoidal vasculopathies.

More preferably, the pathology involving neovascularization is an ocular pathology selected from AMD and choroidal neovascularization.

In a preferred embodiment of the invention, the pathology involving neovascularization is neovascular AMD. This is indeed a group of patients likely to be treated thanks to the anti-angiogenic activity of the factor H fragments according to the invention demonstrated by the inventors.

In a preferred embodiment of the invention, the pathology involving neovascularization is choroidal neovascularization. secondary to AMD or choroidal neovascularization secondary to severe myopia or intraocular inflammation or choroiditis.

"Age-related macular degeneration" or "AMD" is a pathology that first affects the center of the retina, the macula, and in fact causes a loss of precise vision. While preserving peripheral vision, which allows people to navigate globally, AMD affects quality of life significantly. It is the leading cause of irreversible central vision loss in industrialized countries. Age is the primary risk factor for AMD. The EPR in the central part of the retina is considered the first target of AMD. Thus, the damage or loss of function of the EPR causes death of the photoreceptors. Various studies have shown that AMD is a complex pathology linked to many factors, including environmental and genetic factors. There are two forms of AMD: the atrophic form and the wet form. The atrophic form, also called geographic atrophy or dry AMD, results in the progressive disappearance of the cells of the EPR, then to that of the photoreceptors located at the level of the macula. The wet form, also called neovascular or exudative, meanwhile, involves a process of angiogenesis and results in a proliferation of new abnormal vessels under the retina. These fragile vessels leak serum, responsible for an uplift of the retina, and / or blood leading to the appearance of retinal hemorrhages.

"Choroidal neovascularization" corresponds to the appearance of choroidal neovessels. It corresponds to a proliferation of abnormal blood vessels under the retina, in the choroidal layer of the eyeball. Abnormal vessels can leak fluids and blood, causing edema and retinal lifting. It is most often translated by:

metamorphopsia (that is to say, disturbances of vision characterized by deformation of the images), a relative or absolute scotoma (ie a permanent gap in the visual field), and

a visual disturbance predominant in near vision.

The diagnosis is often confirmed by examinations consisting of a fundus of the eye, fluorescein angiography and optical coherence tomography. Choroidal neovascularization may be choroidal neovascularization secondary to AMD or choroidal neovascularization secondary to severe myopia, intraocular inflammation or infectious or non-infectious choroiditis.

MAC formation resulting from activation of the complement pathway has been shown to be responsible for the development of laser-induced choroidal neovascularization in mice (Andreoli et al., 2009). Indeed, the MAC would allow the release of angiogenic factors such as VEGF, FGF-2 and PDGF, resulting in the amplification of the angiogenic process (Andreoli et al., 2009). MAC has also been shown to control VEGF expression in the mouse CNV model (Liu et al., 2011).

In a preferred embodiment, the invention relates to an amino acid sequence selected from the sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO. 10 and SEQ ID No. 12 for the treatment of a pathology selected from AMD, preferably neovascular AMD, choroidal neovascularization and cancer. All the technical characteristics defined previously apply here.

The fragment of the FH according to the invention may be used alone, in combination and / or in combination with other active agents, such as other substances used in the treatment of ocular pathologies, in particular anti-VEGF agents, glucocorticoids, or biological agents such as an anti-factor D antibody. These different active agents can be used in therapeutic combination, and administered in separate, combined, spread or concomitant form. The fragment of FH according to the invention can also be used to prepare a pharmaceutical composition for the treatment and / or prevention of an ocular pathology involving neovascularization. Thus, the invention also relates to a pharmaceutical composition for use in the treatment and / or prevention of an ocular pathology selected from AMD, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, ischemic retinopathy, radiation retinopathy, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, venous vascular occlusion, non-tumor proliferative vasculopathy and cancer. All the technical features mentioned above are applicable here.

The fragments of the FH according to the invention may be combined with any type of pharmaceutically acceptable vehicle or excipient, and optionally with a prolonged diffusion matrix, such as a biodegradable polymer, a biodegradable or non-biodegradable reservoir, particulate systems or implants intraocular or periocular to form a pharmaceutical composition according to the invention. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other undesired reaction when administered to a mammal, particularly a human. A pharmaceutically acceptable carrier or excipient may be solid, semi-solid or liquid. The form of the pharmaceutical compositions, their route of administration, their dosage and their dosage naturally depend on the severity of the ocular pathology and its stage of evolution.

The pharmaceutical composition according to the invention may be in any form that can be administered to a patient, and in particular includes liquid or solid forms, typically in the form of eye drops, gel, or oral solution, but also suspensions, lyophilized powders, capsules and tablets. Formulations formulated so that they can be administered are preferred. locally, such as eye drops and gels, or orally such as drinkable solutions, tablets, ampoules. The pharmaceutical composition according to the invention may also be in a form compatible with an injection, ie a local injection, a mucosal administration, an inhalation, an oral administration and more generally any formulation appropriate for the intended purpose.

Preferably, the pharmaceutical composition according to the invention is in a compatible form for ocular, intraocular, intravitreous, sub-tenonial, subretinal, intra-orbital, subconjunctival, intravenous, intra-ocular administration. arterial, intramuscular, intraperitoneal, topical, subcutaneous, intranasal, oral or parenteral.

The pharmaceutical composition according to the invention comprises from 10% to 90% by weight of a fragment of the FH relative to the total weight of the composition. Preferably, the pharmaceutical composition according to the invention contains from 20 to 60% of a fragment of the FH relative to the total weight of the composition.

Among the modes of administration of a therapeutic protein within the eye, intravitreous injection is a simple way to release a protein in the posterior segment of the eye to minimize systemic exposure. However, repeated injection can lead to complications such as haemorrhage, retinal detachments, or cataracts.

In order to reduce the number of injections and to maintain the activity of the therapeutic protein at a constant level, other modes of administration have been devised. In particular, particulate or non-particulate polymers and particulate lipids (liposomes, micelles, nanoscale emulsions) allow the gradual release of the therapeutic protein that is encapsulated, which reduces the toxicity and increases its shelf life. Finally, the pharmaceutical composition of the invention further comprises another active agent, such as, for example, other substances used in the treatment of ocular pathologies. Typically, this other active agent may be present in the pharmaceutical composition in a proportion of at least 20% by weight relative to the total weight of said composition.

The Factor H fragment according to the invention can be administered in the form of DNA or cDNA for local gene expression. Also, another object of the invention is a polynucleotide comprising the nucleic acid sequence shown in SEQ ID NO: 3 for use in the treatment and / or prevention of a pathology involving neovascularization.

In a preferred embodiment, said polynucleotide comprises a nucleic acid sequence selected from the group consisting of the sequences shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.

Another subject of the invention is an expression vector comprising the polynucleotide encoding the factor H fragment of the invention, or an expression cassette comprising said polynucleotide for its use in the treatment and / or prevention of a pathology involving neovascularization.

According to the invention, the expression vectors suitable for use according to the invention may comprise at least one expression control element operably linked to the nucleic acid sequence. The expression control elements are inserted into the vector and make it possible to regulate the expression of the nucleic acid sequence. Examples of expression control elements include lac systems, lambda phage promoter, yeast promoters or viral promoters. Other operational elements may be incorporated, such as a leader sequence, termination codons, polyadenylation signals and sequences necessary for transcription and subsequent translation of the nucleic acid sequence into the host system. It will be understood by those skilled in the art that the correct combination of expression control elements depends on the chosen host system. It will also be understood that the expression vector must contain the additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Such vectors are easily constructed using conventional or commercially available methods.

The expression of the polynucleotide of the invention may, for example, be carried out locally by any method of transferring the vectorized polynucleotide by viral or non-viral methods into any cell of the eye. The invention therefore relates to a composition comprising a polynucleotide according to the invention for use as a gene therapy medicament. The means by which the vector carrying the polynucleotide can be introduced into the cells include microinjection, electroporation, transduction or transfection using DEAE-dextran, lipofection, calcium phosphate or other procedures. known to those skilled in the art.

In a preferred embodiment, eukaryotic expression vectors that function in eukaryotic cells are used. Examples of such vectors include viral vectors such as retroviruses, adenovirus, herpes virus, vaccinia virus, variola virus, poliovirus, lentivirus, bacterial expression vectors or plasmids such as pcDNA5.

Viral vectors offer the advantage of good transfection efficiency but do not control the amount of secreted proteins. The encapsulated cell technology has been developed to deliver a therapeutic protein in a controlled, continuous, long-term manner in the posterior segment of the eye. In addition, electroporation techniques for specific transfection of the ocular ciliary muscle make it possible to use the ciliary muscle as a reservoir of plasmids encoding therapeutic proteins and thus to express and secrete the fragment according to the invention in intraocular and during an extended duration.

In another aspect, the invention relates to a therapeutic method for the treatment of a pathology involving neovascularization, said method comprising administering a fragment of factor H comprising the amino acid sequence shown in SEQ ID NO: 2. All the technical features mentioned above are applicable here.

In another aspect, the invention relates to the use of a fragment of factor H comprising the amino acid sequence shown in SEQ ID NO: 2 for obtaining a medicament for treating a pathology involving neovascularization. All the technical features mentioned above are applicable here. The following examples are given for illustrative purposes only, and can not limit the scope of the invention. EXAMPLE

The inventors have here demonstrated the therapeutic potential of fragments of FH in relevant models of pathologies involving neovascularization. For this, they produced in cells in culture several fragments of FH. They then proceeded to the purification of these fragments. The assay of the biological activity of the fragments was tested in vitro on the ability of FH (1) fragments to inhibit lysis of sheep erythrocytes induced by the alternative complement pathway (C5b9 complex-related activity), (2) to accelerate the dissociation of the previously formed C3 convertase complex in 96-well ELISA plates (anti-C3 convertase activity), (3) to inactivate, in vitro, the C3b complement molecule by acting as a factor I (FI) cofactor. Finally, the inventors measured the inhibition of angiogenesis by the fragments produced in the model of induction of CNV by laser in the rat. This study revealed for the first time and unexpectedly, an anti-angiogenic role of fragments of FH.

I. Preparation of H factor fragments

The inventors have developed fragments of the FH comprising various domains. the sequence of SCR domains 1 to 7 of human FH, as represented in SEQ ID No. 4, and encoded by the nucleotide sequence SEQ ID No. 5;

the sequence of the SCR domains 1 to 18 of human FH, as represented in SEQ ID No. 6, and encoded by the nucleotide sequence SEQ ID No. 7;

the sequence of 7 to 18 SCR domains of human FH as shown in SEQ ID NO: 10 and encoded by the nucleotide sequence SEQ ID N ⁰ 11,

the sequence of the SCR domains 6 to 8 of human FH, as represented in SEQ ID No. 12, and encoded by the nucleotide sequence SEQ ID No. 13.

the sequence of SCR domains 1 to 4 of human FH, as represented in SEQ ID No. 14 and encoded by the nucleotide sequence SEQ ID No. 15;

the sequence of SCR domains 1 to 6 of human FH, as represented in SEQ ID No. 16 and encoded by the nucleotide sequence SEQ ID No. 17;

the sequence of the SCR domains 8 to 20 of human FH, as represented in SEQ ID No. 18 and encoded by the nucleotide sequence SEQ ID No. 19;

The name, nature and sequence of these fragments are summarized in Table 1 below:

II. Production and purification of H factor fragments

The nucleic sequences of the FH fragments were cloned in a plasmid vector allowing the expression of these molecules in eukaryotic cells. The secretion of these molecules in the cell culture supernatant is favored by the N-terminal use of a judiciously selected signal peptide (MB7). The production of the fragments is carried out by transient transfection in the human cell line HEK 293F. After 7 days of production in batch mode, the supernatant is harvested, clarified, concentrated and sterile filtered (0.22μιη).

The purification of the FH fragments is carried out by affinity chromatography on a Ni-NTA column or cobalt column by means of the hexahistidine tag added at the C-terminal position of the FH fragments. The Shard FH 1NT18, which does not contain a C-terminal tag, was purified by one or two stages of ion exchange chromatography (SP-Sepharose and Q-Sepharose). The eluted FH fragments were dialyzed against PBS buffer and concentrated if the final absorbance concentration at 280 nm was less than 30 ° C / ml.

III. In vitro characterization of functional activity of FH fragments

Material and methods

The anti-C3 convertase activity, expressed in EC50, determines the concentration of FH or FH fragment required to dissociate 50% of the preformed C3bBb3 (IC3) C3 convertase after 32 min incubation at 34 ° C. In a first step, the generation of C3 convertase is therefore carried out in the wells of a microtiter plate as follows: 100 μl of a solution of C3b (2.5 μg / ml) are deposited per well and incubated overnight at 4 ° C; after saturation of the unoccupied binding sites, the step of generating C3 convertase is carried out by adding 4 μg / ml of factor B (FB), 0.3 μg / ml of factor D (FD) and 1.5 mM NiC12 and incubating the mixture at 34 ° C for 120 minutes. In a second step, the formed C3 convertase is dissociated by adding whole FH or FH fragments at different concentrations for 32 to 34 min at 34 ° C. After washing, the amount of the C3 convertase complex still present is determined by detection of human FB using a goat anti-FB antibody and a goat anti-Ig (H + L) antibody coupled to peroxidase.

FH, in the fluid phase, acts as an IF cofactor that cleaves C3b. IF breaks the 110 kDa chain of C3b releasing a 43 kDa fragment and thus produces C3bi. The FH or fragments thereof are incubated in different amounts with purified Factor I and C3b (0.14 μg and 10 μg, respectively) at 37 ° C for 30 minutes. The reaction is stopped by adding denaturation / reduction buffer containing SDS and incubation for 3 minutes at 95 ° C. Control reaction mixtures containing either C3b or C3b and F1 are also prepared and incubated under the same conditions. The samples are then deposited on 10% polyacrylamide gel in the presence of SDS and subjected to electrophoresis (SDS-PAGE). After staining the Coomassie blue gels, the colored bands corresponding to the cleavage products of the α chain are scanned, quantified by densitometric analysis and normalized by the values obtained for the uncleaved chain a '.

The red blood cells of sheep whose surface is rich in sialic acid, bind the FH by its C-terminal part, thus preventing the formation of C3 convertase and consequently cell lysis. A suspension of sheep red blood cells (1x10 GRM / ml) is taken up in a reaction buffer containing 10 mM EGTA and 7 mM MgCl 2 and placed in the presence of a mixture of normal plasma and depleted FH plasma in order to allow the Alternate complement pathway activation. Increasing concentrations of FH or FH fragments are added prior to induction of alternative complement pathway activation to evaluate the inhibition of GRM lysis by these molecules. The mixture is incubated at 37 ° C. in a bath for 30 to 35 minutes, then the reaction is stopped by adding a 10 mM Hepes buffer containing 2 mM EDTA. After centrifugation for 5 minutes at 1730 g, 200 μl of supernatants are taken for an optical density measurement at 414 nm. The measurement of anti-haemolytic activity is determined as a% of the control, the value of which corresponds to the molar concentration of plasma FH necessary to obtain complete inhibition of the lysis (100%).

Results Table 2: Biological Activities of FH Fragments

The results presented in the table above show that:

only fragments containing SCRs 1 to 4 have anti-C3 convertase activity. In addition, the activity of the minimal fragment "1NT4" is significantly lower than that of the fragments containing the following SCRs (SCRs 5 to 18).

similarly, only fragments containing SCRs 1 to 4 have IF cofactor activity.

anti-haemolytic activity is carried by fragments which consist of SCRs 1 to 4 to inhibit C3-convertase activity and SCR 19-20 to attach to sialic acid residues present on the surfaces of sheep red blood cells .

IV. Analysis of the inhibition of angiogenesis

The inventors evaluated the anti-angiogenic activity induced by each of the fragments of the FH described in point I. To do this, they used an in vivo model of choroidal neovascularization (CNV) in Long Evans rats (Janvier Labs, Le Genest -Saint-Isle, France). Material and methods 1. Quantitative test for the determination of choroidal neovascularization.

The rats were anesthetized (using a mixture of ketamine / xylazine in a ratio of ΙΟΟμΙ / 100mg) and the eyes were dilated with mydriaticum and anesthetized with tetracaine. Six Argon laser strikes located at two papillae of the optic nerve were performed at regular intervals. Then, the cornea of these rats was protected with the application of a "Goniosol" gel. Then, 3μ1 of a saline solution alone or a solution of FH diluted in saline solution were injected into the vitreous eyes of each rat. Two injection times were tested: 1- in preventive mode on the same day of the laser and 2- in healing, ie 4 days post-laser. After sacrifice on days 7 or 14 post-laser, rats' eyes were enucleated and then fixed in a solution of paraformaldehyde. Only the posterior part of the eye (EPR / choroid / sclera) was removed and then cut radially. After blocking nonspecific sites with goat serum solution, the EPR / choroid / sclera complexes were labeled with an endothelial cell marker diluted in saline. The EPR / choroid / sclera complexes are then mounted between blade and coverslip using a mounting medium. The markings were observed using a confocal "Zeiss" microscope and images of the entire laser impact, called Stacks, were taken. These images were then analyzed using the "ImageJ" software so that the marking surface was quantified for each impact and for each rat eye. For each experiment, at least 3 groups of 4 rats were tested: 1 non-injected rats (control group), 2 rats injected with saline solution (injection test group) and 3 rats injected with a solution of F H.

Results

The inventors measured the inhibition of choroidal neovascularization conferred by the various fragments of FH produced. These results are summarized in Table 3 below: SCR name of FH Sequence Inhibition of Inhibition of the fragment comprised in acid neovascularization MAC formation the choroidal amino fragment

FH whole SCR 1 to 20 SEQ ID No. 1 76% 85%

"1NT7" SCR 1 to 7 SEQ ID No. 4 73% 69%

"1NT18" SCR 1 to 18, SEQ ID No. 6 67% 59%

"7CT20" SCR 7 to 20 SEQ ID NO: 8 80% 63%

"7CT18" SCR 7 to 18 SEQ ID No. 10 56% 88%

"SCR6-8" SCR 6 to 8 SEQ ID No. 12 0% 85%

"SCR1-4" SCR 1 to 4 SEQ ID No. 14 0% 15%

"SCR1-6" SCR 1 to 6 SEQ ID No. 16 47% 40%

"8CT20 SCR 8 to 20 SEQ ID No. 18 0% 0%

These results clearly demonstrate that the fragments according to the invention, that is to say the fragments comprising the SCR7 domain of FH, allow maximum inhibition of choroidal neovascularization.

Fragments exhibiting inhibition of choroidal neovascularization also act by inhibiting MAC formation.

To the inventors' knowledge, these are the first tests showing an anti-angiogenic role of the fragments of the FH comprising the SCR7 domain.

These fragments then appear to be particularly relevant for the development of strategies for treating pathologies involving neovascularization.

V- Altered Gene Signature of CNV by FH Fragments

To evaluate the activity of rFH and its fragments on the CNV gene signature, a quantitative analysis of the expression of the genes coding for the angiogenesis molecules in the retina complex / pigment epithelium / choroidal cells was performed by the qRT-PCR method.

Results The results show that the rFH1NT7 and rFH7CT20 fragments have the same inhibitory activity of the transcription of angiogenesis genes namely VEGFA, VEGFR1 and VEGFR2 as the entire rFH. In addition, the whole FH and the 2 fragments induce an increase in the expression of the gene coding for PEDF, an angiogenesis inhibitory factor.

Claims

claims

1. Fragment of Factor H for use in the treatment and / or prevention of pathology involving neovascularization, said Factor H fragment comprising the amino acid sequence shown in SEQ ID NO: 2, fused with N domains -terminal or C-terminal of Factor H.

2. Factor H fragment for its use according to claim 1, said fragment comprising the amino acid sequence represented in SEQ ID No. 2 fused with SCR domains 1 to 4 of human factor H and / or SCR domains 19 to 20 of the factor H.

3. Fragment of Factor H for use according to claim 1, said fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID No. 10.

4. Fragment of Factor H for use according to any one of claims 1 to 3, characterized in that said pathology involving neovascularization is selected from the group consisting of AMD, neovascularization of the iris, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, ischemic retinopathy, radiation retinopathy, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, venous vascular occlusion, and non-tumor proliferative vasculopathies and cancer.

5. Factor H fragment for use according to claim 4, characterized in that said cancer is selected from the group consisting of vascularized and / or vascular intraocular tumors, proliferative tumor vasculopathies, vascular tumors of vascular origin or not that choroidal hemangiomas, intraretinal angiomatous proliferations, chorioretinal anastomoses, or polypoidal vasculopathies.

6. Factor H fragment for use according to any one of claims 1 to 4, characterized in that the pathology involving neovascularization is an ocular pathology selected from the group consisting of:

AMD of neovascular form,

choroidal neovascularization, and

- ocular tumors of vascular origin or not.

7. Fragment of Factor H for use according to any one of claims 2 to 4, characterized in that the pathology involving neovascularization is choroidal neovascularization secondary to AMD or choroidal neovascularization secondary to strong myopia, inflammation intraocular or infectious or non-infectious choroiditis.

A pharmaceutical composition comprising a Factor H fragment comprising the amino acid sequence shown in SEQ ID NO: 2, fused with the N-terminal or C-terminal domains of Factor H for use in the treatment and / or prevention ocular pathology selected from AMD, neovascularization of the iris, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, ischemic retinopathy, radiation retinopathy, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, venous vascular occlusion, non-tumor proliferative vasculopathies and cancer.

A pharmaceutical composition comprising a Factor H fragment comprising the amino acid sequence shown in SEQ ID No. 2, fused with the N-terminal or C-terminal domains of Factor H for use according to claim 8, wherein said cancer is selected from the group consisting of vascularized and / or vascular intraocular tumors, proliferative tumoral vasculopathies, vascular tumors of vascular or non-vascular origin such as choroidal hemangiomas, intraretinal angiomatous proliferations, chorioretinal anastomoses or polypoidal vasculopathies.

A polynucleotide comprising the nucleic acid sequence shown in SEQ ID NO: 3 for use in the treatment and / or prevention of a pathology involving neovascularization.

Polynucleotide for use according to claim 10, characterized in that said polynucleotide comprises a nucleic acid sequence selected from the group consisting of the sequences shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: ll.

An expression vector comprising the polynucleotide comprising the nucleic acid sequence shown in SEQ ID NO: 3 for use in the treatment and / or prevention of a pathology involving neovascularization.

A composition comprising a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 3 for use as a gene therapy medicament.