Agents that activate CD47 and their use in treating inflammation

Abstract

To provide agents activating CD47, to provide their use in the treatment of inflammation, in particular treatment-resistant low-grade inflammation, characterized by chronic MP infiltration, such as age-related macular degeneration, and to provide pharmaceutical compositions, medicaments, and kits comprising the agents.SOLUTION: Provided is a multimeric peptide or polypeptide comprising at least two peptide monomers linked through a linker, the at least two peptide monomers activating CD47.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to agents that activate CD47. The present invention also relates to the treatment of inflammatory disorders and diseases, such as age-related macular degeneration. [Background technology]

[0002] Age-related macular degeneration (AMD) is the leading cause of legal blindness in developed countries. Late-stage AMD has two clinical forms: a rapidly progressive exudative form ("wet" AMD) characterized by choroidal neovascularization (CNV), and a slower progressive atrophic form (GA or late-stage "dry" AMD) characterized by retinal pigment epithelium (RPE) atrophy and photoreceptor cell degeneration, known as geographic atrophy. AMD is often classified as "dry" or "wet," but both types develop against a background of increased innate immune activation and are associated with the same genetic polymorphisms, such as complement factor H (CFH) (Haines et al., Science. 2005, 308:419-421; Edwards et al., Science. 2005, 308:421-424), high-temperature-requiring serine protease A1 (HTAR1), and age-related maculopathy susceptibility 2 (ARMSD2) (Dewan et al., Science. 2006, 314:989-992; Yang et al., Science. 2006, 314:992-993).

[0003] Mononuclear phagocytes (MPs) comprise a cell family that includes microglia (MCs), monocytes (Mo), and macrophages (Mφ). Physiologically, MCs are present only in the inner retina. The subretinal space, located between the retinal pigment epithelium (RPE) and photoreceptor outer segments (POS), is an immune-privileged area mediated by immunosuppressive RPE signals, including the leukocyte inhibitor FasL (CD95L). Nevertheless, MPs accumulate in the subretinal space in two vision-threatening forms of advanced AMD (Klein et al., Am J Ophthalmol. 2004, 137:486-495). MPs are in close contact with the RPE in choroidal neovascularization and present at the periphery of RPE lesions in geographic atrophy (Gupta et al., Exp Eye Res. 2003, 76:463-471; Sennlaub et al., EMBO Mol Med. 2013, 5:1775-1793). MPs are thought to contribute to CNV (Tsutsumi et al., J Leukoc Biol. 2003, 74:25-32) and photoreceptor cell degeneration in GA (Cruz-Guilloty et al., Int J Inflam. 2013, 2013, 503-725). Recently, subretinal MPs, an important risk factor for the development of late-stage AMD, have been shown to be present within and around soft drusen (Sennlaub et al., EMBO Mol Med. 2013, 5:1775-1793; Levy et al., EMBO Mol Med. 2015, 7:211-226). However, the causes of altered subretinal immunosuppression and resulting MP accumulation in AMD remain unclear.

[0004] Known treatments for wet AMD include the use of anti-angiogenic agents and photodynamic therapy (laser irradiation of the macula). Anti-angiogenic agents for treating wet AMD include agents that block the action of vascular endothelial growth factor (VEGF), thereby slowing angiogenesis (the formation of new blood vessels in the retina), which causes choroidal neovascularization and vision loss in wet AMD patients. Such "anti-VEGF" agents that have been approved for the treatment of wet AMD or are in clinical trials include bevacizumab (AVASTIN™), ranibizumab (LUCENTIS™), and aflibercept (EYLEA™). New treatment proposals are described, for example, in International Publication No. 2008008986, which discloses the administration of CFHR1 and / or CFHR3 polypeptides. International Patent Application WO 2011137363 relates to treating age-related macular degeneration by reducing 5-lipoxygenase (5-LO) activity. Another example is WO 2014060517, which relates to treating AMD by administering RdCVFL polynucleotides or polypeptides to a subject.

[0005] While there are currently no drugs on the market to treat dry AMD or geographic atrophy, it has been suggested that vitamin supplements containing high doses of antioxidants, lutein, and zeaxanthin may slow progression (Seddon et al., Eye Disease Case-Control Study Group JAMA. 1994, 272:1413-1420). Nonsteroidal anti-inflammatory drugs (NSAIDs) such as glucocorticoids and cyclooxygenase inhibitors, and immunosuppressants such as cyclosporine, are often referred to as "anti-inflammatory" drugs because they inhibit various aspects of inflammation. However, they do not inhibit inflammation overall. Cyclosporine inhibits calcineurin-induced transcription of cytokine genes, primarily in activated T cells, affecting lymphocyte function (Matsuda and Koyasu, 2000), but simultaneously upregulates Toll-like receptors on Mφ (cyclosporine) (Tedesco and Haragsim, Journal of Transplantation. 2012, volume 2012, 230386). Glucocorticoids affect carbohydrate, fat, and protein metabolism and are perhaps best known for their ability to suppress delayed-type hypersensitivity responses by acting directly on T cells (Liu et al., Allergy Asthma Clin Immunol. 2013, 9:30), although at certain concentrations they can exert opposing effects on Mφ function (Lim et al., Immunology. 2007, 122:47-53). NSAIDs are cyclooxygenase inhibitors that inhibit prostaglandin production but increase the synthesis of leukotrienes (Robinson, Clin Exp Rheumatol. 1989, 7 Suppl 3:S155-161), which activate MPs (Gagnon et al., Agent Actions. 1989, 26:141-147) and may perpetuate MP infiltration (Gilroy et al., Nat Med. 1999, 5:698-701). This lack of efficacy in inhibiting MP-mediated subretinal inflammation may explain why widely used "anti-inflammatory" therapies, such as systemic NSAID treatment, have not slowed AMD progression.

[0006] Taken together, these findings suggest that 'anti-inflammatory' therapies specifically tailored to inhibit the mechanisms of subretinal MP accumulation and activation in AMD are needed.

[0007] AMD is associated with treatment-resistant and low-grade chronic inflammation that primarily involves the innate immune system and, most notably, the accumulation of MPs (Combadiere et al., J Clin Invest. 2007, 117:2920-2928; Levy et al., EMBO Mol Med. 2015, 7:211-226). In contrast to rapidly progressing autoimmune lesions characterized by cytotoxic T lymphocytes, neutrophils, and MPs (Caspi, International reviews of immunology. 2002, 21:197-208; Kerr et al., Prog Retin Eye Res. 2008, 27:527-535), the infiltrating leukocytes in slowly progressing GA are primarily MPs, as are other long-term age-related diseases, including atherosclerosis, neurodegenerative diseases, and cancer (Grivennikov et al., Cell. 2010, 140:883-899; Hotamisligil, Cell. 2010, 140:900-917). More generally, inflammation is the organism's response to tissue injury and microbial invasion. Ideally, inflammation rapidly and efficiently eliminates pathogens and repairs tissue damage through either regeneration or scarring. If the inflammatory response is not promptly controlled, it can become pathogenic and contribute to disease progression, as seen in many chronic inflammatory diseases. Treatment-resistant and low-grade chronic inflammation is seen in conditions such as metabolic diseases (obesity, atherosclerosis) (Hotamisligil, Cell. 2010, 140:900-917), neurodegenerative diseases (Glass et al., Cell. 2010, 140:918-934), and cancer (Grivennikov et al., Cell. 2010, 140:883-899), and thus contributes significantly to the pathogenesis of many chronic age-related diseases. While treatment-resistant inflammation is not the primary cause of these diseases, it contributes significantly to their pathogenesis because the bactericidal mediators (e.g., reactive oxygen species, proteases, and inflammatory cytokines) produced by neutrophils and interstitial macrophages can also cause significant secondary damage to host cells, which can themselves lead to more inflammation. It is often unclear how long chronic inflammation persists, either because of a primary problem of persistence or because of an inability to break the cycle of inflammation, secondary damage, and new inflammation.Affected tissues are often associated with the presence of mononuclear phagocytes (MPs), a cell family that includes resident macrophages (rMφ) such as monocytes (Mo), microglial cells, and monocyte-derived inflammatory macrophages (iMφ) that arise during inflammation, but are rarely associated with lymphocyte infiltration or adaptive immune responses (Nathan and Ding, Cell. 2010, 140:871-882).

[0008] Therefore, in view of the above factors, there remains a need to identify active ingredients for the prevention and / or treatment of treatment-resistant low-grade inflammation, more particularly inflammation associated with the accumulation of mononuclear phagocytes, and even more particularly AMD.

[0009] This object is achieved by the present invention, since the inventors have surprisingly shown that TSP1 mediates mononuclear phagocyte elimination via its receptor CD47.

[0010] CD47 is known to play an important role in immune and angiogenic responses. In particular, binding of TSP-1 to CD47 affects multiple fundamental cellular functions, including cell migration and adhesion, cell proliferation, and apoptosis, and plays a role in regulating angiogenesis and lymphocyte clearance (Chao et al., Curr. Opin. Immunol. 2012, 24(2):225-32). CD47 also interacts with signal-regulatory protein alpha (SIRPα), an inhibitory transmembrane receptor present on myeloid cells. CD47 / SIRPα interaction results in bidirectional signal transduction, resulting in distinct intercellular responses, including inhibition of phagocytosis, stimulation of cell-cell fusion, and T cell activation (Barclay, Curr. Opin. Immunol. 2009, 21(1):47-52).

[0011] International Patent Application WO 99 / 40940 discloses the use of anti-CD47 antibodies for the prevention or treatment of inflammatory diseases. Similarly, WO 2010 / 70047 describes CD47-binding polypeptides for use as drugs in the treatment of autoimmune and inflammatory disorders. WO 2011 / 143624 also discloses the use of anti-CD47 antibodies to regulate phagocytosis. However, the treatment of inflammatory diseases according to these patent applications necessarily involves the inhibition of CD47.

[0012] Surprisingly, the present inventors have demonstrated that CD47 activation is crucial for mononuclear phagocyte elimination and that CD47 activation is mediated by its ligand, TSP1. Furthermore, the present inventors have shown that HTRA1 degrades TSP1, thereby inhibiting CD47 activation and mononuclear phagocyte elimination. Furthermore, the present inventors have surprisingly demonstrated that combined therapy with a CD47 agonist and a Fas agonist reverses HTRA1-induced mononuclear phagocyte accumulation, thereby treating inflammation.

[0013] Thus, the present invention relates to agents that activate CD47 and their use in treating treatment-resistant MP accumulation and inflammation, such as age-related macular degeneration. Summary of the Invention

[0014] The present invention relates to agents that activate CD47 for use in the treatment of inflammation.

[0015] In one embodiment, the agent for use in treating inflammation of the present invention directly activates CD47. In one embodiment, the agent is a CD47 agonist, preferably a TSP1 peptide mimetic. In another embodiment, the agent is an activating peptide selected from the group including 4N1K, PKHB1, and PKT16.

[0016] In another embodiment, the agent for use in treating inflammation of the present invention indirectly activates CD47. In one embodiment, the agent is selected from the group comprising a TSP1 activator, an HTRA1 inhibitor, and a Fas activator.

[0017] In one embodiment, the inflammation according to the present invention is acute inflammation or chronic inflammation. In one embodiment, the agent is for use in treating treatment-resistant chronic inflammation, preferably treatment-resistant low-grade inflammation. In one embodiment, the inflammation is inflammation associated with the accumulation of mononuclear phagocytes. In one embodiment, the inflammation is selected from the group consisting of age-related macular degeneration; retinitis pigmentosa; neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, or Alzheimer's disease; and metabolic disorders such as obesity or atherosclerosis. In a specific embodiment, the inflammation is age-related macular degeneration.

[0018] The present invention further relates to compositions comprising at least one of the above-mentioned agents. In one embodiment, a composition according to the invention comprises a CD47 agonist and a Fas activator.

[0019] Another object of the present invention is a pharmaceutical composition comprising at least one agent that activates CD47 and at least one pharmaceutically acceptable carrier for use in the treatment of inflammation, preferably age-related macular degeneration.

[0020] A further object of the present invention is a medicament comprising at least one agent that activates CD47 for use in the treatment of inflammation, preferably age-related macular degeneration.

[0021] In one embodiment, the agent, pharmaceutical composition or medicament is administered intraocularly, preferably by intravitreal injection, or applied by topical intraocular administration.

[0022] The present invention also relates to kits comprising at least one of the above-mentioned agents, pharmaceutical compositions or medicaments.

[0023] definition For the purposes of the present invention, the following terms have the following meanings:

[0024] The term "amino acid" is understood to include the 20 naturally occurring amino acids; those amino acids that are often post-translationally modified in vivo, such as hydroxyproline, phosphoserine, and phosphothreonine; and other unusual amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Furthermore, in one embodiment, the term "amino acid" includes both D- and L-amino acids (stereoisomers).

[0025] The term "amino acid substitution" refers to the replacement of one amino acid with another amino acid in a polypeptide. In one embodiment, an amino acid is replaced with another amino acid with similar structural and / or chemical properties, e.g., a conservative amino acid substitution. "Conservative amino acid substitutions" can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Non-conservative substitutions involve exchanging a member of one of the above classes for another. For example, amino acid substitutions may replace one amino acid with another amino acid that differs in structure and / or chemical properties, e.g., replacing an amino acid from one group (e.g., polar) with another amino acid from a different group (e.g., basic). Amino acid substitutions can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods other than genetic engineering, such as methods that alter the side chain group of an amino acid by chemical modification, may also be useful.

[0026] The term "identity" refers to a measure of the identity of nucleotide or amino acid sequences. Generally, sequences are aligned to obtain the highest order match. "Identity" itself has an art-recognized meaning and can be calculated using published techniques. See, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, von Heijne, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991. Although there are several methods for measuring the identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those skilled in the art (Carillo and Lipton, SIAM J Applied Math, 1998, 48:1073). Commonly used methods for determining identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, edited by Martin J. Bishop, Academic Press, San Diego, 1994; and Carillo and Lipton, SIAM J Applied Math, 1998, 48:1073. Methods for determining identity and similarity have been codified in the form of computer programs.A preferred computer program method for determining identity and similarity between two sequences includes, but is not limited to, the GCG program package (Devereux et al., J Molec Biol, 1990, 215:403). Most preferably, the program used to determine identity levels was the GAP program used in the examples below.

[0027] By way of example, a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain an average of up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted or substituted with other nucleotides, or several nucleotides may be inserted into the reference sequence up to 5% of the total nucleotides of the reference sequence. Such mutations of the reference sequence may be present at the 5' or 3' end of the reference nucleotide sequence, or anywhere between these end positions, interspersed individually among the nucleotides of the reference sequence, or in one or more contiguous groups within the reference sequence.

[0028] The term "peptide" refers to a linear polymer of amino acids consisting of fewer than 50 amino acids joined together by peptide bonds. The peptides of the present invention are not limited to a particular length product. This term does not refer to and excludes post-expression modifications of peptides, both naturally occurring and non-naturally occurring, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art.

[0029] The term "peptide linker," also referred to as a "spacer peptide," refers to a peptide used to link two peptides or polypeptides together. In one embodiment, a peptide linker of the present invention comprises 3 to 50 amino acids. Peptide linkers are known in the art or described herein. In one embodiment of the present invention, a peptide linker is also referred to as "L."

[0030] The term "polynucleotide" refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-stranded and double-stranded RNA and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or may be a mixture of single- and double-stranded regions. Furthermore, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also encompasses DNA or RNA containing one or more modified bases and DNA or RNA whose backbones have been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and rare bases, such as inosine. DNA and RNA are variously modified, and thus "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short polynucleotides, often referred to as oligonucleotides.

[0031] The term "polypeptide" refers to any peptide or protein containing two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly called peptides, oligopeptides, or oligomers, and to longer chains, generally called proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.

[0032] The term "protein" refers to a sequence and / or multimeric entity of 100 or more amino acids. Proteins of the present invention are not limited to products of a particular length. The terms "polypeptide" or "protein" do not refer to or exclude post-expression modifications of proteins, both naturally occurring and non-naturally occurring, such as glycosylation, acetylation, phosphorylation, and other modifications known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as a voluminous research literature. Modifications can occur anywhere in a polypeptide or protein, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. It will be understood that the same type of modification can be present in the same or varying degrees at multiple sites in a given polypeptide or protein. A given polypeptide or protein can also contain multiple types of modifications. Polypeptides or proteins can be branched as a result of ubiquitination and can be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides or proteins can result from post-transcriptional natural processes or can be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of amino acids to proteins via transfer RNA, such as arginylation, and ubiquitination.See, for example, "Proteins - structure and molecular properties," 2nd ed., TECreighton, W.H. Freeman and Company, New York, 1993; Wolt, F., "Posttranslational Protein Modifications: Perspectives and Prospects," in Posttranslational covalent modification of proteins, BC Johnson (ed.), Academic Press, New York, 1983, pp. 1-12; Seifter et al., "Analysis for protein modifications and nonprotein cofactors," Meth Enzymol, 1990, 182:626-646; Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging," Ann NY Acad Sci, 1992, 663:48-62. A protein can be an entire protein or a subsequence thereof. An "isolated protein" is a protein that has been identified and separated and / or recovered from a component of its natural environment.

[0033] In a preferred embodiment, the isolated protein comprises: (1) greater than 80%, 85%, 90%, 95%, and most preferably greater than 96%, 97%, 98%, or 99% by weight of protein, as determined by the Lowry method; (2) to an extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) SDS-PAGE to homogeneity under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Refine.

[0034] Isolated protein includes protein in situ within recombinant cells since at least one component of the protein's natural environment will not be present. Ordinarily, however, isolated protein will be prepared by at least one purification step.

[0035] The term "function-conservative fragment" refers to a peptide derived from a peptide of the present invention in which a given amino acid residue has been altered without altering the overall conformation and function of the peptide, including, but not limited to, substitution of an amino acid with an amino acid of similar properties (e.g., polarity, hydrogen-bonding ability, acidic, basic, hydrophobic, aromatic, etc.). Because amino acids other than those described as conserved within a given protein may differ, the percent protein or amino acid sequence similarity between any two functionally similar proteins may vary, for example, from 70% to 99% when similarity is determined by alignment methods such as the Cluster method based on the MEG ALIGN algorithm. "Function-conservative variants" also encompass polypeptides that have at least 20%, preferably 40%, more preferably 60%, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90% amino acid identity, as determined by the BLAST or FASTA algorithms, and that have the same or substantially the same properties or functions as the native or parent protein to which they are compared.

[0036] The term "derivative" refers to a variant of the polypeptide of the invention or a function-conserving variant thereof that has been modified in another way, i.e., by the addition of a compound to any amino acid of the sequence, by covalently attaching any type of molecule to the polypeptide, in order to modify the conformation, activity, specificity, effect or stability of the polypeptide in vitro or in vivo.

[0037] The term "agonist" refers to a natural or synthetic compound that binds to a protein and stimulates the biological activity of that protein, thereby stimulating the effect of said protein. Consequently, a "CD47 agonist" includes any chemical entity that, when administered to a subject, results in stimulation of a biological activity associated with CD47 in the patient, including any downstream biological effect that would otherwise result from the binding of CD47 to its natural ligand. Such CD47 agonists include any agent capable of stimulating CD47 expression or any downstream biological effect of CD47.

[0038] The term "immunoglobulin" encompasses polypeptides having a combination of two heavy chains and two light chains, regardless of whether they have any significant specific immunoreactivity. The term "antibody" refers to a combination of two heavy chains and two light chains that have significant known specific immunoreactivity against a desired antigen (e.g., CD47, TSP1, HTRA1, or Fas). Antibodies and immunoglobulins comprise light and heavy chains, whether or not there is a covalent interchain bond between them. The basic immunoglobulin structure in vertebrate systems is relatively well understood. The collective term "immunoglobulin" encompasses five different classes of antibodies that can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention, and the following description generally refers to immunoglobulin molecules of the IgG class. With regard to IgG, immunoglobulins comprise two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000 to 70,000 daltons. The four chains are connected by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, which begin at the mouth of the "Y" and continue throughout the variable region. Antibody light chains are classified as kappa or lambda ([κ], [λ]). Each class of heavy chain can be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently linked to each other, and the "tail" regions of the two heavy chains are linked to each other by covalent disulfide bonds or, if the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, by noncovalent bonds. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will recognize that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4). It is the nature of these chains that qualifies an antibody as a "class": IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes will be readily apparent to those skilled in the art in light of the present disclosure and are therefore within the scope of the present invention. As described above, the variable region of an antibody enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and VH domain of an antibody combine to form the variable region, which defines a three-dimensional antigen-binding site. This antibody quaternary structure forms the antigen-binding site present at the end of each arm of the Y-shape. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) of each of the VH chain and VL chain.

[0039] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., a population in which the individual antibodies within the population are identical except for minor natural mutations that may be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include various antibodies directed against different determinants (epitopes), monoclonal antibodies each are directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be produced using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0040] The term "polyclonal antibody" refers to a population of immunoglobulin molecules that react against a specific antigen and each recognize a different epitope. Thus, in contrast to monoclonal antibodies, polyclonal antibodies are not derived from a single cell line.

[0041] The term "antibody fragment" refers to a portion or region of an antibody that contains fewer amino acid residues than an intact or complete antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with the intact antibody (i.e., the intact antibody from which it is derived) for antigen binding (i.e., specific binding to CD47). As used herein, the term "antibody fragment" of an antibody molecule encompasses antigen-binding fragments of antibodies, such as antibody light chain variable domains (VL), single-chain antibody heavy chain variable domains (VH), single-chain antibodies (scFv), F(ab')2 fragments, Fab fragments, Fd fragments, Fv fragments, single-domain antibody fragments (Dab), mono-armed (monovalent) antibodies, diabodies, triabodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, or any antigen-binding molecule formed by combining, assembling, or conjugating such antigen-binding fragments. Fragments can be obtained, for example, by chemical or enzymatic treatment of an intact or complete antibody or antibody chain, or by recombinant means.

[0042] The term "derived from" preceding a specified protein (e.g., a TSP1 antibody or antigen-binding fragment thereof) refers to the origin of the polypeptide. In one embodiment, a polypeptide or amino acid sequence derived from a particular starting polypeptide is a CDR sequence or a sequence related thereto. In one embodiment, an amino acid sequence derived from a particular starting polypeptide is not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are derived from a single starting antibody. In one embodiment, a polypeptide or amino acid sequence derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence substantially identical to the sequence of the starting sequence, or a region of at least 3-5 amino acids, 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids thereof, or a region whose origin is identifiable to one of skill in the art as being in the starting sequence.

[0043] The term "diabody" refers to small antibody fragments prepared by constructing sFv fragments (see paragraph on sFv) using a short linker (about 5-10 residues) between the VH and VL domains such that interchain, rather than intrachain, V domain pairing occurs, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers composed of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP 404,097; WO 93 / 11161; and Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448 (1993).

[0044] The term "peptibody": A peptibody consists of a biologically active peptide grafted onto an Fc domain. In this way, certain desirable characteristics of antibodies are retained, in particular the apparent increase in affinity due to the avidity that results from the dimerization of two Fc domains.

[0045] The term "epitope" refers to a specific amino acid arrangement present on a peptide or protein(s) to which an agent (e.g., an antibody or small molecule) binds. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear or conformational, i.e., they can include two or more sequences of amino acids from different regions of an antigen that are not necessarily contiguous.

[0046] The term "Fv" refers to the minimum antibody fragment that contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. Folding of these two domains results in six hypervariable regions (three loops from each of the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0047] The terms "immunospecific," "specific for," or "specifically binds to" as used herein mean that the antibody specifically binds to a substance at a detectable level, preferably about 10 4 M -1 That's about 10 5 M -1 That's about 10 6 M -1 That's about 10 7 M -1 That's it, 10 8 M -1 That's it, 10 9 M -1 More than or equal to 10 10 M -1 An antibody is said to be "immunospecific," "specific for," or "specifically binds to" an antigen if it reacts with the antigen with an affinity constant Ka equal to or greater than 10. The affinity of an antibody for its cognate antigen is often expressed as a dissociation constant, Kd, ​​and in certain embodiments, an antibody may have a dissociation constant Kd of 10 or greater.-4 M or less, about 10 -5 M or less, about 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 5.10 -9 M or less, 10 -9 M or less, 5.10 -10 M or less or 10 -10 It specifically binds to an antigen if it binds with a Kd of less than or equal to M. The affinity of an antibody can be readily determined using conventional techniques, such as those described in Scatchard G et al. (Ann NY Acad Sci. 1949, 51:660-672). The binding properties of an antibody to an antigen, cell, or tissue can generally be determined and evaluated using immunodetection methods, including immunofluorescence-based assays such as immunohistochemistry (IHC) and / or fluorescence-activated cell sorting (FACS).

[0048] The term "mammal" refers to any mammal, including humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a human.

[0049] The term "synthetic" with respect to polypeptides includes polypeptides that contain amino acid sequences that do not occur in nature. For example, a non-naturally occurring polypeptide is a modified form of a naturally occurring polypeptide (e.g., one that contains mutations such as additions, substitutions, or deletions) or a polypeptide that contains a first amino acid sequence (whether naturally occurring or not) linked to a second amino acid sequence (whether naturally occurring or not) that is not naturally linked to it in the linear sequence of amino acids.

[0050] The term "small molecule" refers to low molecular weight molecules, including lipids, simple sugars, second messengers, and other natural products and metabolites. Small molecules are distinct from macromolecules such as proteins.

[0051] The term "binding site" includes a region of a polypeptide that is involved in selective binding to a target antigen of interest (e.g., CD47, TSP1, HTRA1, or Fas). A binding domain or binding region comprises at least one binding site. Exemplary binding domains include antibody variable domains. An antibody molecule of the invention can comprise a single antigen-binding site or multiple (e.g., two, three, or four) antigen-binding sites.

[0052] The term "siRNA" or "small interfering RNA" refers to a double-stranded structure containing about 15 to about 50 base pairs, e.g., about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to a target gene or RNA expressed in a cell. siRNAs comprise a sense RNA strand and a complementary antisense RNA strand annealed to each other through standard Watson-Crick base-pairing interactions. The sense strand comprises a nucleic acid sequence substantially identical to a nucleic acid sequence contained within a target miRNA molecule. "Substantially identical" to a target sequence contained within a target mRNA refers to a nucleic acid sequence that differs from the target sequence by about 3% or less. The sense and antisense strands of an siRNA may comprise two complementary single-stranded RNA molecules, or may comprise a single molecule in which the two complementary portions are base-paired and covalently linked by a single-stranded "hairpin" region. siRNAs can be produced chemically or biologically, or expressed from recombinant plasmids or viral vectors, using methods well known to those of skill in the art.

[0053] The term "antisense oligonucleotide" (or "ASO") refers to a small deoxyribonucleotide with a sequence complementary to the mRNA of a target gene. Such oligonucleotides bind to the target mRNA through complementary base pairing and attract the binding of RNase H, an enzyme that degrades double-stranded RNA, thereby destroying the target mRNA.

[0054] The term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures; the goal is to prevent or slow (reduce) the rate of inflammation. Those in need of treatment include those already with inflammation and those who are prone to inflammation or in whom inflammation is to be prevented. A subject or mammal has been successfully "treated" of inflammation if, after administration of a therapeutic amount of an agent according to the invention, the patient experiences an observable and / or measurable reduction or absence of one or more of the following: some alleviation of one or more symptoms associated with inflammation, a reduction in morbidity and mortality, and an improvement in quality of life. The above parameters for assessing successful treatment and improvement of disease are readily measured by routine methods familiar to physicians.

[0055] The term "subject" refers to a mammal, preferably a human. In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting or receiving medical treatment, or having been / is / will be the subject of medical treatment, or being monitored for the development of inflammation. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the compound of the invention is administered to a human patient in need thereof.

[0056] The term "therapeutically effective amount" refers to a level or amount of an agent that (1) delays or prevents the onset of inflammation; (2) delays or halts the progression, worsening, or deterioration of one or more symptoms of inflammation; (3) brings about improvement of the symptoms of inflammation; (4) reduces the severity or frequency of inflammation; or (5) cures inflammation, without causing significant negative or harmful side effects to the target. For prophylactic or preventative treatment, a therapeutically effective amount may be administered before the onset of inflammation. Alternatively, or in addition, a therapeutically effective amount may be administered after the onset of inflammation for therapeutic or maintenance treatment.

[0057] The term "pharmaceutically acceptable excipient" refers to an excipient that does not cause adverse, allergic, or other untoward reactions when administered to animals, preferably humans. The term includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semisolid, or liquid fillers, diluents, encapsulating materials, or formulation auxiliary agents of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

[0058] The term "about" before a numerical value means 10% greater or less than said numerical value. DETAILED DESCRIPTION OF THE INVENTION

[0059] (Detailed explanation) The present invention relates to a multimeric peptide or polypeptide comprising at least two peptide monomers linked to each other, wherein said at least two peptide monomers activate CD47.

[0060] In one embodiment, a multimeric peptide or polypeptide of the invention comprises amino acids of the 4N1K peptide (sequence KRFYVVMWKK, SEQ ID NO: 1), the PKHB1 peptide (sequence (D)KRFYVVMWK-(D)K, Formula I) and / or the PKT16 peptide (sequence (D)K-(NMeR)-FYVV-Nle-WK-(D)K, Formula II), or a functionally conserved fragment thereof.

[0061] [ka]

[0062] In one embodiment, the multimeric peptide or polypeptide comprises at least 5 consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 1 and function-conservative fragments thereof. In another embodiment, the agent of the embodiment, an activated polypeptide or protein of the invention comprises at least 6, 7, 8, 9, or 10 consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 1 and function-conservative fragments thereof.

[0063] In one embodiment, the at least two peptide monomers are the same or different. For example, in one embodiment, a multimeric peptide or polypeptide may comprise two 4N1K peptides. As a further example, in another embodiment, a multimeric peptide or polypeptide may comprise one 4N1K peptide and one PKT16 peptide.

[0064] In one embodiment, a multimeric peptide or polypeptide of the present invention comprises at least two 4N1K peptides or functionally conservative fragments thereof. In one embodiment, a multimeric peptide or polypeptide of the present invention comprises at least two PKHB1 peptides or functionally conservative fragments thereof. In one embodiment, a multimeric peptide or polypeptide of the present invention comprises at least two PKT16 peptides or functionally conservative fragments thereof.

[0065] In one embodiment, a multimeric peptide or polypeptide of the invention comprises at least one 4N1K peptide and at least one PKHB1 peptide or a functionally conserved fragment thereof. In one embodiment, a multimeric peptide or polypeptide of the invention comprises at least one 4N1K peptide and at least one PKT16 peptide or a functionally conserved fragment thereof. In one embodiment, a multimeric peptide or polypeptide of the invention comprises at least one PKHB1 peptide and at least one PKT16 peptide or a functionally conserved fragment thereof.

[0066] In one embodiment, a multimeric peptide or polypeptide of the invention comprises any number of repeating units. In one embodiment, a multimeric peptide or polypeptide of the invention comprises 2 to 10, 2 to 20, or 2 to 30 repeating subunits. In one embodiment, a multimeric peptide or polypeptide of the invention comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 repeating subunits. Thus, in one embodiment, a multimeric peptide or polypeptide of the invention can be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, eleven-mer, or duodecamer.

[0067] In certain embodiments, the multimeric peptides or polypeptides of the present invention comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 4N1K peptides. Thus, in certain embodiments, the multimeric peptides or polypeptides of the present invention are 4N1K peptide dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, eleven-mers, or duodecamers. In certain embodiments, the multimeric peptides or polypeptides of the present invention are 4N1K dimers.

[0068] In one embodiment, conjugation of the peptide monomers of the present invention can be carried out using any method known in the art, so long as it does not substantially interfere with the biological activity of the multimeric peptide, polypeptide or protein, i.e., activation of CD47.

[0069] In one embodiment, the peptide monomers of the present invention may be linked via a linking moiety.

[0070] Examples of linking moieties include, but are not limited to, simple covalent bonds, flexible peptide linkers, alkyl linkers, disulfide bridges, or polymers such as polyethylene glycol (PEG). Peptide linkers can be entirely artificial (e.g., containing 2 to 20 amino acid residues independently selected from the group consisting of glycine, serine, asparagine, threonine, and alanine) or derived from natural proteins. Disulfide bridge formation can be achieved, for example, by the addition of cysteine ​​residues, as further described herein below. Linkage via polyethylene glycol (PEG) can be achieved by reacting a monomer having a free cysteine ​​with a multifunctional PEG, such as linear bis-maleimide PEG. Alternatively, linkage can be performed via glycans on the monomer after oxidation to the aldehyde form and using a multifunctional PEG containing an aldehyde-reactive group. The location of the linkage between two monomers should be selected so that the linkage does not substantially interfere with the ability of the multimeric peptide or polypeptide to activate CD47.

[0071] In one embodiment, the linking moiety is a peptide linker.

[0072] In one embodiment, the peptide linker of the present invention is 3 to 30 amino acids in length, preferably 4 to 20 amino acids, and more preferably 5 to 15 amino acids in length. In one embodiment, the peptide linker of the present invention contains at least 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 amino acids. In one embodiment, the peptide linker of the present invention contains up to 20, 19, 18, 17, 16, 15, 14, 13, or 12 amino acids.

[0073] In one embodiment, a peptide linker of the invention comprises 3, 4, 5, 6, 7, 8, or 9 amino acids, hi another embodiment, a peptide linker of the invention comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids.

[0074] Examples of peptide linkers include, but are not limited to, Gly-rich linkers such as poly-Gly linkers, Ser-rich linkers, linkers containing a stretch of Gly and Ser residues (also called "GS linkers"), Pro-rich linkers, helix linkers, and the like.

[0075] In one embodiment, the amino acids of the peptide linker are selected from the 20 naturally occurring amino acids. In a preferred embodiment, 1 to 20 amino acids are selected from Gly, Ala, Pro, Asn, Gln, Cys, and Lys. In a more preferred embodiment, the linker is a sterically unhindered amino acid, such as Gly, Gly-Gly [(Gly)2], Gly-Gly-Gly [(Gly)3]...(Gly). 20 , Ala, Gly-Ala, Ala-Gly, Ala-Ala, etc. Other specific examples of linkers are (Gly)3Lys(Gly)4 (SEQ ID NO: 2); (Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 3) (this structure provides a glycosylation site when recombinantly produced in a mammalian cell line capable of glycosylation); (Gly)3Cys(Gly)4 (SEQ ID NO: 4); and GlyProAsnGly (SEQ ID NO: 5).

[0076] In a preferred embodiment, the peptide linker is Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly [(Gly)8, SEQ ID NO: 6]. In another preferred embodiment, the peptide linker is a combination of Gly and Ala. In another preferred embodiment, the peptide linker is a combination of Gly and Lys.

[0077] In one embodiment, the multimeric peptide or polypeptide of the present invention comprises two 4N1K peptides linked via a peptide linker, preferably a Gly-rich linker. In another embodiment, the multimeric peptide or polypeptide of the present invention comprises two PKHB1 peptides linked via a peptide linker, preferably a Gly-rich linker. In one embodiment, the multimeric peptide or polypeptide of the present invention comprises two PKT16 peptides linked via a peptide linker, preferably a Gly-rich linker.

[0078] In a particular embodiment, the multimeric peptide or polypeptide of the invention comprises or consists of the amino acid sequence of SEQ ID NO:7.

[0079] The present invention also relates to polynucleotide or nucleic acid sequences that encode the multimeric peptides or polypeptides described herein above.

[0080] In one embodiment, the polynucleotide or nucleic acid is DNA. In another embodiment, the polynucleotide of the present invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0081] Another object of the present invention is a vector comprising one or more polynucleotides encoding the multimeric peptides or polypeptides of the present invention. In a preferred embodiment, the vector of the present invention is an expression vector.

[0082] A further object of the present invention is a composition comprising a multimeric peptide or polypeptide of the invention as described herein above.

[0083] Another object of the present invention is a modified TSP1 protein that is resistant to the protease HTRA1 (HTRA1-resistant modified TSP1) or a fragment thereof, wherein said modified TSP1 protein activates CD47.

[0084] Applicants show that HTRA1 cleaves TSP1 at (i) a site known to bind to integrin α3β1, (ii) two sites between the "type 2" domains, and (iii) two sites between two valine-valine-methionine (VVM) sequences that can interact with the CD47 receptor and are responsible for its efficient activation (see Example 4). In contrast, TSP2, a protein with the same overall structure and that interacts with several of the same cell surface receptors, including CD47, is resistant to the protease HTRA1.

[0085] In one embodiment, a fragment of a modified TSP1 protein that is resistant to the protease HTRA1 comprises 50 to 1,100, 100 to 1,000, 900, 800, 700, 600, 500, or 400 amino acids of the modified TSP1 protein. In another embodiment, a fragment of the present invention comprises 150 to 1,100, 1,000, 900, 800, 700, 600, 500, or 400 amino acids of the modified TSP1 protein. In another embodiment, a fragment of the present invention comprises 200 to 1,100, 1,000, 900, 800, 700, 600, 500, or 400 amino acids of the modified TSP1 protein. In another embodiment, a fragment of the invention comprises 300 to 1100, 1000, 900, 800, 700, 600, 500, or 400 amino acids of the modified TSP1 protein. In a specific embodiment, a fragment of the invention comprises 369 amino acids of the modified TSP1 protein.

[0086] In one embodiment, a fragment of the invention comprises or consists of the C-terminal portion of a modified TSP1 protein. In a preferred embodiment, a fragment of the invention comprises or consists of the last 369 amino acids of a modified TSP1 protein.

[0087] In one embodiment, the modified TSP1 protein or fragment thereof retains the ability to bind to CD47 while being resistant to the protease HTRA1.

[0088] In one embodiment, the HTRA1-resistant modified TSP1 of the present invention is a modified TSP1 protein in which at least one amino acid in at least one of the HTRA1 cleavage sequences has been deleted, substituted, or added.

[0089] As used herein, "HTRA1 cleavage sequence" refers to a sequence within the amino acid sequence of TSP1 that is cleaved by the protease HTRA1. In one embodiment, at least one HTRA1 cleavage sequence of TSP1 is QVTQ at positions 241-244 of SEQ ID NO:8. In one embodiment, at least one HTRA1 cleavage sequence of TSP1 is GQVR at positions 287-290 of SEQ ID NO:8.

[0090] In one embodiment, the HTRA1-resistant modified TSP1 of the present invention is a modified TSP1 protein in which at least one amino acid is deleted from at least one of the HTRA1 cleavage sequences.

[0091] In one embodiment, the HTRA1-resistant modified TSP1 of the present invention is a modified TSP1 protein in which residues VT at positions 242-243 of SEQ ID NO: 8 are deleted. In one embodiment, the HTRA1-resistant modified TSP1 of the present invention is a modified TSP1 protein in which residues QV at positions 288-289 of SEQ ID NO: 8 are deleted. In one embodiment, the HTRA1-resistant modified TSP1 of the present invention is a modified TSP1 protein in which residues VT at positions 242-243 and residues QV at positions 288-289 of SEQ ID NO: 8 are deleted.

[0092] In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or fragment thereof of the invention comprises a sequence at least 75% identical to SEQ ID NO: 9. In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or fragment thereof of the invention comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 9. In one embodiment, the HTRA1-resistant modified TSP1 or fragment thereof of the invention has an amino acid sequence comprising or consisting of SEQ ID NO: 9. In one embodiment, the HTRA1-resistant modified TSP1 of the invention has a C-terminal portion having the amino acid sequence of SEQ ID NO: 9. In one embodiment, the HTRA1-resistant modified TSP1 fragment of the invention has an amino acid sequence consisting of SEQ ID NO: 9.

[0093] In one embodiment, the HTRA1-resistant modified TSP1 of the present invention is a modified TSP1 protein in which at least one amino acid in at least one of the HTRA1 cleavage sequences has been substituted.

[0094] In one embodiment, the HTRA1-resistant modified TSP1 of the invention is a modified TSP1 protein in which residue V at position 242 of SEQ ID NO: 8 has been substituted. In one embodiment, the HTRA1-resistant modified TSP1 of the invention is a modified TSP1 protein in which residue V at position 289 of SEQ ID NO: 8 has been substituted. In one embodiment, the HTRA1-resistant modified TSP1 of the invention is a modified TSP1 protein in which residues V242 and V289 of SEQ ID NO: 8 have been substituted.

[0095] In one embodiment, the substitution is an amino acid substitution selected from the group consisting of A, C, D, F, G, H, I, L, M, N, P, Q, R, S, V, W, and Y. In a particular embodiment, the substitution is amino acid N. In one embodiment, the HTRA1-resistant modified TSP1 or fragment thereof is a modified TSP1 protein comprising the substitution V242N (position according to SEQ ID NO: 8). In another embodiment, the HTRA1-resistant modified TSP1 or fragment thereof is a modified TSP1 protein comprising the substitution V289N (position according to SEQ ID NO: 8). In another embodiment, the HTRA1-resistant modified TSP1 or fragment thereof is a modified TSP1 protein comprising the substitutions V242N and V289N (positions according to SEQ ID NO: 8).

[0096] In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or a fragment thereof comprises a sequence at least 75% identical to SEQ ID NO: 11. In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or a fragment thereof comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 11. In one embodiment, the HTRA1-resistant modified TSP1 or a fragment thereof comprises an amino acid sequence comprising or consisting of SEQ ID NO: 11. In one embodiment, the HTRA1-resistant modified TSP1 of the invention has a C-terminal portion having the amino acid sequence of SEQ ID NO: 11. In one embodiment, the HTRA1-resistant modified TSP1 fragment of the invention has an amino acid sequence consisting of SEQ ID NO: 11.

[0097] In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or a fragment thereof further comprises at least one cysteine ​​substitution. In one embodiment, the at least one substituted cysteine ​​is selected from the group consisting of C3, C15, C34, C35, C55, C73, C93, C109, C129, C145, C191, and C366 (positions according to SEQ ID NO: 8). In one embodiment, the at least one substituted cysteine ​​is C34 (position according to SEQ ID NO: 8). In another embodiment, the at least one substituted cysteine ​​is C191 (position according to SEQ ID NO: 8). In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or a fragment thereof further comprises two cysteine ​​substitutions. In one embodiment, the two substituted cysteines are C34 and C191 (positions according to SEQ ID NO: 8).

[0098] In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or fragment thereof of the invention comprises a sequence at least 75% identical to SEQ ID NO: 10. In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or fragment thereof of the invention comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 10. In one embodiment, the HTRA1-resistant modified TSP1 or fragment thereof of the invention has an amino acid sequence comprising or consisting of SEQ ID NO: 10. In one embodiment, the HTRA1-resistant modified TSP1 of the invention has a C-terminal portion having the amino acid sequence of SEQ ID NO: 10. In one embodiment, the HTRA1-resistant modified TSP1 fragment of the invention has an amino acid sequence consisting of SEQ ID NO: 10.

[0099] In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or a fragment thereof comprises a sequence at least 75% identical to SEQ ID NO: 12. In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 or a fragment thereof comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 12. In one embodiment, the HTRA1-resistant modified TSP1 or a fragment thereof has an amino acid sequence comprising or consisting of SEQ ID NO: 12. In one embodiment, the HTRA1-resistant modified TSP1 of the present invention has a C-terminal portion having the amino acid sequence of SEQ ID NO: 12. In one embodiment, the HTRA1-resistant modified TSP1 fragment has an amino acid sequence consisting of SEQ ID NO: 12.

[0100] In one embodiment, the HTRA1-resistant modified TSP1 or fragment thereof is a modified TSP1 protein or fragment thereof in which an HTRA1 cleavage sequence replaces the HTRA1-resistant sequence of TSP2.

[0101] In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 comprises a sequence that is at least 75% identical to SEQ ID NO: 13. In one embodiment, the amino acid sequence of the HTRA1-resistant modified TSP1 comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 13. In one embodiment, the HTRA1-resistant modified TSP1 has an amino acid sequence that comprises or consists of SEQ ID NO: 13.

[0102] In one embodiment, the HTRA1-resistant derivative TSP2 of the invention is a chimeric TSP2 / TSP1 recombinant protein comprising (i) the amino acid sequence of TSP2 and (ii) the amino acid sequence of a TSP1 tail, wherein the TSP1 tail comprises a second VVM sequence of TSP1. In one embodiment, the amino acid sequence of the TSP1 tail comprising the second VVM sequence is located at the N-terminus of the chimeric TSP2 / TSP1 recombinant protein.

[0103] In one embodiment, an HTRA1-resistant derived TSP2 of the invention comprises a sequence at least 75% identical to SEQ ID NO: 14. In one embodiment, an HTRA1-resistant derived TSP2 of the invention comprises a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 14. In one embodiment, an HTRA1-resistant derived TSP2 of the invention has an amino acid sequence comprising or consisting of SEQ ID NO: 14.

[0104] The present invention also relates to polynucleotide or nucleic acid sequences that encode modified TSP1 proteins or fragments thereof that are resistant to the protease HTRA1 as described herein above.

[0105] In one embodiment, the polynucleotide or nucleic acid is DNA. In another embodiment, the polynucleotide of the present invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0106] Another object of the present invention is a vector comprising one or more polynucleotides encoding a modified TSP1 protein or fragment thereof that is resistant to the protease HTRA1 according to the present invention. In a preferred embodiment, the vector of the present invention is an expression vector.

[0107] A further object of the present invention is a composition comprising a modified TSP1 protein or a fragment thereof or a polynucleotide that is resistant to the protease HTRA1 as described above.

[0108] In one embodiment, the multimeric peptide or polypeptide of the present invention or the modified TSP1 protein or fragment thereof that is resistant to the protease HTRA1 according to the present invention has a modification that increases the stability of the peptide, polypeptide or protein in the body or increases its ability to penetrate into cells.

[0109] Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH, or CF=CH, backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are described, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[0110] In one embodiment, the peptide bond (-CO-NH-) in the peptide may be replaced by, for example, an N-methylated bond (-N(CH3)-CO-), an ester bond (-C(R)HCOOC(R)-N-), a ketomethylene bond (-CO-CH2-), an α-aza bond (-NH-N(R)-CO-) (where R is any alkyl, e.g., methyl), a carba bond (-CH2-NH-), a hydroxyethylene bond (-CH(OH)-CH2-), a thioamide bond (-CS-NH-), an olefinic double bond (-CH=CH-), a reverse amide bond (-NH-CO-), a peptide derivative (-N(R)-CH2-CO-) (where R is a "normal" side chain naturally occurring on a carbon atom).

[0111] In one embodiment, these modifications can be present at any bond along the peptide chain, possibly at multiple bonds (2-3) at the same time.

[0112] In one embodiment, synthetic, unnatural acids such as phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe, or o-methyl-Tyr may be substituted for the natural aromatic amino acids Trp, Tyr, and Phe.

[0113] In one embodiment, the peptides, polypeptides, or proteins of the present invention may be linear or cyclic. "Cyclic" means that at least two separate, i.e., non-contiguous, portions of the molecule are linked to one another. For example, the amino and carboxy termini of the molecule may be covalently linked to form a cyclic molecule. Alternatively, the molecule may contain two or more Cys residues (e.g., within a linker) and be cyclized via disulfide bond formation. Furthermore, it is contemplated that two or more tandem peptide dimers may combine to form a dimer of dimers. Thus, for example, a tandem dimer containing a Cys residue may form an intermolecular disulfide bond with the Cys residue of another such dimer.

[0114] In one embodiment, the peptides, polypeptides or proteins of the invention may be covalently or non-covalently bound to a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched polymer; a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide.

[0115] Other possible carriers include the addition of one or more water-soluble polymers, such as polyoxyethylene glycol or polypropylene glycol. Additional useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of these polymers.

[0116] In a preferred embodiment, the carrier is polyethylene glycol (PEG). In one embodiment, the PEG group can be of any convenient molecular weight and can be linear or branched. In one embodiment, the average molecular weight of the PEG ranges from about 2 kDa to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, and most preferably from about 5 kDa to about 10 kDa.

[0117] In one embodiment, the PEG group is attached to the compound of the invention via acylation, reductive alkylation, Michael addition, thiol alkylation, or other chemoselective conjugation / ligation methods via a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, ct-haloacetyl, maleimide, or hydrazino group) to a reactive group on the target compound (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimide, or hydrazino group).

[0118] In one embodiment, carbohydrate (oligosaccharide) groups are attached to known glycosylation sites within a protein. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues, and N-linked oligosaccharides are attached to asparagine (Asn) residues as part of the sequence Asn-X-Ser / Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids except proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One common type of sugar found in both is N-acetylneuraminic acid (called sialic acid). Sialic acid is typically the terminal residue of both N-linked and O-linked oligosaccharides, and its negative charge can impart acidic properties to glycosylated compounds. Such site(s) may be incorporated into the linker of the compounds of the invention and are preferably glycosylated by the cell during recombinant production of the polypeptide compound (e.g., in mammalian cells such as CHO, BHK, COS, etc.), although such sites may also be further glycosylated by synthetic or semi-synthetic methods known in the art.

[0119] In one embodiment, the above peptides, polypeptides or proteins may be further fused to one or more Fc polypeptides, either directly or via a linker group.

[0120] In one embodiment, the Fc sequence of the compound can be selected from the human immunoglobulin IgG-1 heavy chain (see Ellison, JW et al., Nucleic Acids Res. 10:4071-4079 (1982)) or any other Fc sequence known in the art (e.g., other IgG classes, including but not limited to IgG-2, IgG-3, and IgG-4 or other immunoglobulins).

[0121] It is well known that the Fc region of an antibody is composed of monomeric polypeptide segments that can be linked by disulfide bonds or non-covalent bonds to form dimers or multimers. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from one to four, depending on the antibody class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2) involved. As used herein, the term "Fc" refers collectively to monomeric, dimeric, and multimeric Fc molecules. It should be noted that Fc monomers will spontaneously dimerize if appropriate Cys residues are present, unless specific conditions exist that prevent dimerization via disulfide bond formation.

[0122] In one embodiment, the Fc polypeptide may be any variant having a longer in vivo half-life than a control Fc polypeptide. Examples of Fc polypeptide variants include, but are not limited to, Fc polypeptides that bind to FcRn with a higher affinity than a control Fc polypeptide at slightly acidic pH, Fc polypeptides that bind to FcRn with an affinity equal to or less than that of a control Fc polypeptide at physiological pH, and Fc polypeptides containing an insertion of 3 to 20 amino acids within or adjacent to loops 5, 8, and / or 10.

[0123] In one embodiment, the protein of the present invention comprises at least one multimeric peptide as described above further fused to one or more Fc groups. In a preferred embodiment, the protein of the present invention comprises at least one multimeric peptide comprising two 4N1K peptides linked via a peptide linker, preferably a Gly-rich linker, further fused to one or more Fc groups.

[0124] In a specific embodiment, the protein of the invention comprises two multimeric peptides comprising two 4N1K peptides linked via a peptide linker, each of the two 4N1K peptides further fused to an Fc polypeptide.

[0125] Applicant provides herein CD47 - / - Mouse, Tsp1 - / - Similar to mice, age-, light-, and laser-induced accumulation of subretinal mononuclear phagocytes occurs, but CD36 - / - (See Example 1.) - / - Microglial cells and CD47 - / - Microglial cells showed significant resistance to elimination compared with wild-type microglial cells, and recombinant TSP1 highly significantly promoted the elimination of wild-type microglial cells, suggesting that Tsp1 - / - The microglial cell phenotype was restored to normal, but CD47 - / -No effect was observed on microglial cells, confirming that the interaction between TSP1 and CD47 mediates microglial cell elimination (see Example 1). Applicant also shows that HTRA1 is highly expressed early in monocyte-macrophage differentiation and that SNP rs11200638 significantly increases HTRA1 expression (see Example 2). Applicant shows that HTRA1 proteolyzes TSP1 at five distinct sites, two of which are located between the two valine-valine-methionine sites required for efficient CD47 activation (see Examples 3 and 4). Furthermore, in vitro, recombinant HTRA1 significantly increased the survival of mononuclear phagocytes cocultured with RPE cells and CD47 activation by activating peptides (see Example 5), and this effect was further enhanced by activating peptides containing two CD47-binding sites (see Example 6). Simultaneous activation of CD47 by TSP-1 and FAS by MegaFasL reversed this effect (see Example 5). In vivo, we demonstrate that intravitreal injection of recombinant TSP-1 or CD47-activating peptides efficiently promoted the removal of subretinal mononuclear monocytes after laser-induced subretinal inflammation in inflammation-prone Cx3cr1-deficient mice (see Example 6). Furthermore, recombinant TSP-1 or CD47-activating peptides efficiently promoted the removal of inflammatory macrophages in a sterile peritonitis model (see Example 6). Taken together, we show that (i) CD47 activation and (ii) the combination of CD47 and FAS activation efficiently eliminate mononuclear phagocytes.

[0126] Thus, the present invention relates to an agent that activates CD47 for use in the treatment of inflammation. In one embodiment, the agent that activates CD47 is used to treat (or is for use to treat) inflammation.

[0127] Within the meaning of the present invention, the term "activate" means that an agent is capable of directly or indirectly activating the biological activity of a target protein. In certain embodiments, an agent that activates CD47 is an agent that is capable of activating the biological activity of CD47 upon directly or indirectly activating CD47.

[0128] In one embodiment, an agent of the invention directly activates CD47. Examples of agents that directly activate CD47 include, but are not limited to, CD47 agonists, activating antibodies, activating peptides, activating polypeptides, activating proteins, peptibodies, and the like.

[0129] As used herein, the term "agonist of CD47" refers to proteins and peptides that are capable of binding to and activating the receptor CD47 to produce its biological activity.

[0130] In one embodiment, the CD47 agonist is its natural ligand, TSP-1, a TSP1 variant, TSP1 fragment, or TSP1 peptidomimetic that binds to CD47 and retains the ability of TSP1 to induce the downstream biological effect of CD47 activation, i.e., elimination of intracellular mononuclear phagocytes.

[0131] In one embodiment, the variant, fragment or peptidomimetic of TSP1 is a modified TSP1 protein that is resistant to the protease HTRA1 (HTRA1-resistant modified TSP1) as specified herein above, or a fragment thereof.

[0132] In another embodiment, the TSP1 variant, fragment or peptidomimetic is a derived TSP2 protein (HTRA1-resistant derived TSP2) as specified herein above that retains the ability to bind to CD47 while being resistant to the protease HTRA1.

[0133] In another embodiment, the agonist of CD47 is its natural ligand SIRPα, a SIRPα variant, a SIRPα fragment or a SIRPα peptidomimetic.

[0134] In one embodiment, the CD47 agonist is an activating antibody. Examples of activating antibodies include, but are not limited to, the antibody Ad22 (Pettersen et al., J. Immunol. 1999, 162(12):7031-40), the antibody 1F7 (Manna et al., J. Biol. Chem. 2005, 280:29637-29644), and the antibody MABL (Uno et al., Oncology Reports. 2007, 17(5):1189-1194).

[0135] In another embodiment, the CD47 agonist is an activating peptide. Examples of activating peptides include, but are not limited to, the 4N1K peptide (SEQ ID NO: 1); the PKHB1 peptide (Formula I); and the PKT16 peptide (Formula II).

[0136] In one embodiment, activation peptides of the present invention are 5 to 15 amino acids, 6 to 14 amino acids, 7 to 13 amino acids, 8 to 12 amino acids, or 9 to 11 amino acids in length. In another embodiment, activation peptides of the present invention are 5 to 14 amino acids, 5 to 13 amino acids, 5 to 12 amino acids, 5 to 11 amino acids, or 5 to 10 amino acids in length. In another embodiment, activation peptides of the present invention are 6 to 15 amino acids, 7 to 15 amino acids, 8 to 15 amino acids, 9 to 15 amino acids, or 10 to 15 amino acids in length.

[0137] In one embodiment, an activation peptide of the invention comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, an activation peptide of the invention comprises at least 5 consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 1 and functionally conserved fragments thereof. In particular, CD47 is selected from the group consisting of TSP1 activators, HTRA1 inhibitors, and Fas activators.

[0138] In another embodiment, an activation peptide of the invention, an agent of the embodiment, comprises at least 6, 7, 8, 9, or 10 consecutive amino acids selected from the amino acid sequence of SEQ ID NO: 1 and functionally conserved fragments thereof. In a preferred embodiment, an activation peptide of the invention consists of the amino acids of SEQ ID NO: 1.

[0139] In one embodiment, the activation peptide of the present invention is selected from the group consisting of 4N1K, PKHB1, PKT16, and functionally conservative fragments thereof. In one embodiment, the activation peptide of the present invention is PKHB1, PKT16, or a functionally conservative fragment thereof. In one embodiment, the activation peptide of the present invention is PKHB1 or a functionally conservative fragment thereof. In another embodiment, the activation peptide of the present invention is PKT16 or a functionally conservative fragment thereof.

[0140] In one embodiment, the CD47 agonist is a multimeric peptide or polypeptide as defined hereinabove. In one embodiment, the CD47 agonist is a multimeric peptide or polypeptide comprising at least one 4N1K peptide. In one embodiment, the multimeric peptide or polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 repeating subunits of the 4N1K peptide. In a specific embodiment, the CD47 agonist is a multimeric peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7. In another embodiment, the agent of the present invention indirectly activates CD47. In one embodiment, the agent of the present invention that indirectly activates CD47 is an HTRA1 inhibitor. Examples of HTRA1 inhibitors include, but are not limited to, antibodies against HTRA1, variants or fragments thereof, siRNAs and antisense oligonucleotides (ASOs) against the HTRA1 gene and / or transcripts of the HTRA1 gene.

[0141] In another embodiment, the agent of the invention is a Fas activator. Examples of Fas activators include, but are not limited to, proteins and peptides capable of binding to Fas and activating DISC formation, as well as variants, fragments, or peptidomimetics of FasL that retain the ability of FasL to bind to Fas and induce apoptosis of the corresponding cells.

[0142] In certain embodiments, the Fas activator of the present invention preferably includes a FasL ligand or any functional fragment or derivative thereof. In one embodiment, the Fas activator used in the present invention is a Fas receptor agonist.

[0143] In certain embodiments, the Fas activator of the present invention preferably includes the Fas receptor agonist APO010 (TopoTarget, Copenhagen, Denmark), a recombinant soluble hexameric fusion protein consisting of three human Fas ligand (FasL) extracellular domains fused to the dimerization collagen domain of human adiponectin, potentially possessing proapoptotic and anti-cancer activity. The Fas receptor agonist APO010 activates the Fas receptor in susceptible tumor cell populations, resulting in caspase-dependent apoptosis (Verbrugge et al., 2009). In certain embodiments, the Fas activator of the present invention preferably includes the Fas agonist MegaFasL (AdipoGen). Additionally, other Fas activators of the present invention preferably include the Fas agonist peptides disclosed in U.S. Patent Nos. 6,001,962 and 6,846,637.

[0144] In one embodiment, an agent of the invention is a prophylactic and / or therapeutic agent, hi a specific embodiment, an agent of the invention is a therapeutic agent.

[0145] Within the meaning of the present invention, "inflammation" is as defined in Dorland's Medical Dictionary as "a local protective response elicited by tissue injury or destruction that destroys, thins, or sequesters both the injurious agent and the injured tissue." Inflammation is characterized by fenestrations of the capillary system, leakage of blood elements into the interstitial space, and migration of leukocytes into the inflamed tissue. At the macroscopic level, inflammation is usually accompanied by familiar clinical signs such as erythema, edema, hyperalgesia (tenderness), and pain.

[0146] In one embodiment the agent according to the invention is for use in the treatment of inflammation, said inflammation being selected from the group comprising age-related macular degeneration (AMD), retinitis pigmentosa, Parkinson's disease, multiple sclerosis, Alzheimer's disease, obesity, atherosclerosis, allergies, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis or psoriatic arthritis), asthma, graft versus host disease, peritonitis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome, systemic lupus erythematosus, nephritis and ulcerative colitis.

[0147] In one embodiment, the inflammation of the present invention is acute inflammation, hi another embodiment, the inflammation of the present invention is chronic inflammation.

[0148] In one embodiment, the inflammation of the present invention is treatment-resistant inflammation. In one embodiment, the inflammation of the present invention is low-grade chronic inflammation. In one embodiment, the inflammation of the present invention is treatment-resistant low-grade inflammation.

[0149] In one embodiment, the inflammation of the present invention is a treatment-resistant low-grade inflammation selected from the group including age-related diseases such as age-related macular degeneration (AMD) and age-related maculopathy (ARM); metabolic diseases such as obesity and atherosclerosis; neurodegenerative diseases and cancer. In one embodiment, the treatment-resistant low-grade inflammation of the present invention is age-related macular degeneration (AMD).

[0150] In one embodiment, the prophylactic and / or therapeutic agents of the invention are for use in treating inflammation associated with mononuclear phagocyte accumulation.

[0151] Mononuclear phagocytes (MPs) comprise a cell family that includes microglial cells (MCs), monocytes (Mo), and macrophages (Mφ). Inflammation associated with mononuclear phagocyte accumulation includes, but is not limited to, retinal inflammation such as age-related macular degeneration (AMD), age-related maculopathy (ARM), or retinitis pigmentosa; neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, or Alzheimer's disease; metabolic disorders such as obesity or atherosclerosis; allergies; ankylosing spondylitis; arthritis such as osteoarthritis, rheumatoid arthritis, or psoriatic arthritis; asthma, graft-versus-host disease; peritonitis, Crohn's disease; colitis; dermatitis; diverticulitis; fibromyalgia; hepatitis; irritable bowel syndrome; systemic lupus erythematosus; nephritis; and ulcerative colitis. In one embodiment, the inflammation according to the present invention is peritonitis.

[0152] In one embodiment, the inflammation according to the present invention is selected from the group comprising retinal inflammation such as age-related macular degeneration (AMD), retinitis pigmentosa or age-related maculopathy; neurodegenerative diseases such as Parkinson's disease, multiple sclerosis or Alzheimer's disease; metabolic disorders such as obesity or atherosclerosis.

[0153] In one embodiment, the inflammation according to the present invention is an age-related disease selected from the group comprising AMD, age-related maculopathy, retinitis pigmentosa, atherosclerosis and neurodegenerative diseases such as Parkinson's disease, multiple sclerosis or Alzheimer's disease.

[0154] In one embodiment, the inflammatory disease of the present invention is not a cancer or a tumor.

[0155] The retina is particularly vulnerable to immunopathogenic damage due to its extremely limited regenerative capacity, yet it is particularly protected from direct infection (sclera, eyelids) as well as blood-borne bacterial invasion (blood-tissue barrier). Furthermore, this tissue is a site of "immune privilege," which contributes to its protection from inflammation-mediated injury. Factors contributing to immune privilege include the lack of lymphatic drainage systems (e.g., in the eye and brain) for DCs and antigen-presenting cells to migrate to lymph nodes, the lack of blood vessels for effector cells to infiltrate tissues (cornea, subretinal space), and locally produced factors that induce immune tolerance. Importantly, this privilege is also mediated by tonic inhibitory signals in the retina that set a higher activation threshold compared to non-immune-privileged tissues, and by the efficient removal of infiltrating inflammatory cells (immunosuppressive microenvironment) (Streilein et al., Vision Res. 2002, 42:487-495). In this way, potential antigen-presenting cells and effector cells (lymphocytes, macrophages) can be neutralized before they can produce cytotoxicity.

[0156] In one embodiment, the inflammation according to the present invention is non-autoimmune inflammation. Examples of autoimmune inflammatory diseases include, but are not limited to, inflammation of the kidney, liver and lungs, atherosclerosis and metabolic syndrome, Behcet's disease and endometriosis.

[0157] In one embodiment, the inflammation according to the present invention is autoimmune inflammation. Examples of autoimmune inflammatory diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, celiac sprue disease, scleroderma, psoriasis, inflammatory bowel disease, and Sjogren's syndrome.

[0158] In one embodiment, the inflammation according to the present invention is ocular inflammation. As used herein, ocular inflammatory disease refers to inflammation occurring in any part of the eye or surrounding tissue. Thus, ocular inflammatory disease is a disease resulting from inflammation in the eye or in the optic nerve, blood vessels, muscles, or other tissues surrounding the eye.

[0159] In one embodiment, the ocular inflammation is selected from the group comprising or consisting of age-related macular degeneration (AMD), retinitis pigmentosa, age-related maculopathy (ARM), uveitis, scleritis, episcleritis, optic neuritis, keratitis, orbital pseudotumor, retinal vasculitis, and chronic conjunctivitis.

[0160] In one embodiment, the inflammation according to the present invention is not an ocular inflammation.

[0161] In one embodiment, the inflammation according to the present invention is retinal inflammation.

[0162] Within the meaning of the present invention, "retinal inflammation" refers to inflammation of the subretinal space mediated by mononuclear phagocytes. In one embodiment, retinal inflammation according to the present invention includes age-related macular degeneration (AMD), age-related maculopathy, and retinitis pigmentosa.

[0163] In one embodiment, the agent of the invention is for use in treating age-related macular degeneration, which in certain embodiments includes atrophic (or dry) AMD and neovascular (or wet) AMD.

[0164] In one embodiment, the prophylactic and / or therapeutic agents of the invention are for use in the treatment of dry AMD, hi another embodiment, the prophylactic and / or therapeutic agents of the invention are for use in the treatment of neovascular AMD.

[0165] In one embodiment, the AMD according to the present invention is in the early stage, characterized by the accumulation of extracellular deposits called drusen in and around the macula, associated with hyperpigmentation (abnormalities of the pigment epithelium).

[0166] In another embodiment, the AMD according to the present invention is in late stage.Late stage is characterized by unilateral or bilateral complications.Late stage AMD can be dry AMD or wet AMD.In certain embodiments, AMD is late stage dry AMD (also called geographic AMD).

[0167] In one embodiment, the agent of the invention is for use in treating age-related maculopathy (ARM). In one embodiment, the ARM is an early ARM. In another embodiment, the ARM is a late ARM.

[0168] In one embodiment, the agent of the invention is for use in the treatment of retinitis pigmentosa.

[0169] In one embodiment, the subject suffers from inflammation, preferably inflammation associated with mononuclear phagocyte accumulation.In certain embodiments, the subject suffers from retinal inflammation.In preferred embodiments, the subject suffers from age-related macular degeneration (AMD), age-related maculopathy (ARM) or retinitis pigmentosa.

[0170] In one embodiment, the subject suffers from early stage AMD. In another embodiment, the subject suffers from late stage AMD. In one embodiment, the subject suffers from choroidal neovascular AMD ("wet" AMD). In another embodiment, the subject suffers from geographic atrophy ("dry" AMD).

[0171] In one embodiment, the subject is suffering from early stage ARM, hi another embodiment, the subject is suffering from late stage ARM.

[0172] In another embodiment, the subject is prone to develop inflammation, i.e. prone to mononuclear phagocyte accumulation.In certain embodiments, the subject is at risk of developing retinal inflammation.In preferred embodiments, the subject is at risk of developing AMD, ARM or retinitis pigmentosa.

[0173] Examples of risks for developing AMD and ARM include, but are not limited to, genetics, lifestyle factors such as smoking, sun exposure or poor diet, age, excessive blood cholesterol levels, high blood pressure, and the like.

[0174] In one embodiment, the subject of the present invention is an elderly subject. As used herein, the term "elderly subject" refers to a subject who is at least 50 years old, at least 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, or 90 years old.

[0175] In certain embodiments, the risk of developing AMD is due to the existence of SNP rs11200638 located in the HTRA1 promoter on human chromosome 10q26.In Japanese and Caucasian populations, SNP rs11200638 is associated with a 10-fold increase in the risk of wet age-related macular degeneration.The genotype with the highest risk is (A;A).

[0176] In one embodiment, the subject has not yet been treated with another therapy for inflammation, preferably AMD, ARM, or retinitis pigmentosa, hi another embodiment, the subject has already been treated with another therapy for inflammation, preferably AMD, ARM, or retinitis pigmentosa.

[0177] The present invention also relates to compositions comprising at least one agent that activates CD47 as described herein above. In one embodiment, the compositions of the present invention comprise at least one agent that activates CD47 for use in the treatment of inflammation.

[0178] In one embodiment, the composition is used (or is intended for use in treating) inflammation.

[0179] In one embodiment, a composition of the invention comprises at least one agent that directly activates CD47 and at least one agent that indirectly activates CD47. In a specific embodiment, a composition of the invention comprises an agent that activates CD47 and an agent that activates Fas.

[0180] Another object of the present invention is a pharmaceutical composition comprising at least one agent of the invention as described herein above and at least one pharmaceutically acceptable excipient.

[0181] Pharmaceutically acceptable additives that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0182] The present invention further relates to a medicament comprising at least one agent, composition or pharmaceutical composition of the present invention.

[0183] In one embodiment, the composition, pharmaceutical composition or medicament of the invention is used (or is intended for use in the treatment of) inflammation, preferably AMD.

[0184] Preferably, the composition, pharmaceutical composition or medicament of the invention comprises a therapeutically effective amount of the agent of the invention.

[0185] In one embodiment, the composition, pharmaceutical composition or medicament of the invention further comprises an additional prophylactic and / or therapeutic agent, which in one embodiment is another agent for treating inflammation, particularly AMD.

[0186] It will be understood that the total daily dosage of the compounds, compositions, pharmaceutical compositions, and medicaments of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific compound used; the duration of treatment; drugs used in combination with or simultaneously with the specific compound used; and similar factors well known in the medical field. For example, it is well within the skill of one of ordinary skill in the art to start with a dose of the compound lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dosage of the product can vary over a wide range, from about 10 to about 10,000 mg per adult per day, preferably from 100 to about 5,000 mg, and more preferably from about 200 to about 2,000 mg per adult per day. Preferably, the composition contains 10 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg, or 2000 mg of the active ingredient, with the dosage for the patient being treated being adjusted to the symptoms. The medicament typically contains about 10 to about 10,000 mg of the active ingredient, preferably 5 to about 5,000 mg, and more preferably about 10 to about 2,000 mg. An effective amount of the drug is typically provided at a dosage level of 0.01 mg to about 100 mg per kg of body weight per day, preferably about 0.05 mg to 40 mg per kg of body weight per day, more preferably about 0.1 mg to 20 mg per kg of body weight per day, and more preferably about 0.2 mg to 1 mg per kg of body weight per day.

[0187] In one embodiment, the therapeutically effective amount is within the range of about 10 to about 10,000 mg / ml, preferably 100 to about 5,000 mg / ml, and more preferably about 200 to about 2,000 mg / ml of the composition, pharmaceutical composition, or medicament of the present invention.

[0188] In one embodiment, the therapeutically effective amount is within the range of about 10 to about 10,000 mg / g, preferably 100 to about 5,000 mg / g, and more preferably about 200 to about 2,000 mg / g of the composition, pharmaceutical composition, or medicament of the present invention.

[0189] In one embodiment, the therapeutically effective amount is within the range of about 10 to about 10,000 mg / ml, preferably 5 to about 5,000 mg / ml, more preferably about 10 to about 2,000 mg / ml, and more preferably about 20 to about 1,000 mg / ml of the composition, pharmaceutical composition, or medicament of the present invention.

[0190] In one embodiment, the therapeutically effective amount is within the range of about 10 to about 10,000 mg / g, preferably 5 to about 5,000 mg / g, more preferably about 10 to about 2,000 mg / g, and more preferably about 20 to about 1,000 mg / g of the composition, pharmaceutical composition, or medicament of the present invention.

[0191] In one embodiment of the present invention, the prophylactic and / or therapeutic agent comprises a CD47 activator at a concentration of about 5 mg / mL to about 500 mg / mL, about 5 mg / mL to about 100 mg / mL, or about 5 mg / mL to about 10 mg / mL.

[0192] In one embodiment of the present invention, the prophylactic and / or therapeutic agent comprises a CD47 activator at a concentration of about 0.1 μM to about 1000 μM, preferably about 1 μM to about 750 μM, more preferably about 5 μM to about 600 μM, and even more preferably about 10 μM to about 500 μM.

[0193] In another embodiment of the present invention, the prophylactic and / or therapeutic agent comprises a CD47 activator at a concentration of about 1 μg / mL to about 1 mg / mL, about 1 μg / mL to about 500 μg / mL, or about 1 μg / mL to about 100 μg / mL.

[0194] In another embodiment of the present invention, the prophylactic and / or therapeutic agent comprises a CD47 activator at an intraocular concentration of about 1 to about 10 μg per mL of human intraocular fluid, preferably about 5 μg per mL of human intraocular fluid.

[0195] For use in administering to a subject, the composition is formulated for administration to a subject.The composition of the present invention can be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, bucally, vaginally, or via an implanted reservoir.The term administration used herein includes subcutaneous, intravenous, intramuscular, intraocular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0196] In one embodiment, the composition, pharmaceutical composition or medicament of the present invention is in a form suitable for oral administration.

[0197] Examples of forms suitable for oral administration include, but are not limited to, tablets, orodispersibles / orodispersible tablets, effervescent tablets, powders, granules, pills (including sugar-coated pills), sugar-coated tablets, capsules (including soft gelatin capsules), syrups, liquids, gels or other drinkable liquids, suspensions, slurries, liposomal forms, and the like.

[0198] In one embodiment, the composition, pharmaceutical composition or medicament of the present invention comprises one or more pharmaceutically acceptable carriers for a formulation suitable for oral administration.

[0199] In one embodiment, the inventive composition, pharmaceutical composition or medicament of the present invention is in a form suitable for topical administration.

[0200] Examples of forms suitable for topical administration include, but are not limited to, liquid, paste or solid compositions, more particularly those in the form of aqueous solutions, drops, eye drops, eye drops, dispersions, sprays, microcapsules, microparticles, nanoparticles, polymer patches or controlled-release patches. In a preferred embodiment, the composition, pharmaceutical composition or medicament of the present invention is in the form of eye drops.

[0201] In one embodiment, the composition, pharmaceutical composition or medicament of the invention is in a form suitable for injection, such as intraocular, intramuscular, subcutaneous, intradermal, transdermal or intravenous injection or infusion.

[0202] Examples of forms suitable for injection include, but are not limited to, liquid forms, such as sterile aqueous solutions, dispersions, emulsions, suspensions, and solid forms suitable for use in preparing solutions or suspensions by adding a liquid prior to use, such as powders, liposomal forms, and the like.

[0203] The compositions of the present invention in sterile injectable form may be aqueous or oily suspensions. These suspensions may be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions using non-toxic diluents or solvents acceptable for parenteral administration. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, conventional methods use sterile, fixed oils as solvents or suspending media. For this purpose, any non-irritating, fixed oil, including synthetic monoglycerides or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectable solutions, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, particularly in polyoxyethylated forms. These oily solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. For formulation purposes, other commonly used surfactants, such as Tween, Span and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms may also be used.

[0204] In a particular embodiment, the composition, pharmaceutical composition or medicament of the present invention is in a form suitable for intraocular administration, preferably for intraocular injection.

[0205] Within the meaning of the present invention, "intraocular administration" refers to the direct injection of an agent into the interior of the eye, where the interior of the eye refers to any region located within the eyeball, and generally includes, but is not limited to, any functional (e.g., visually relevant) or structural tissue found within the eyeball, or any tissue or cell layer lining part or all of the interior of the eyeball. Specific examples of such regions include the anterior chamber, posterior chamber, vitreous cavity, choroid, macula, and retina, as well as the blood vessels and nerves vascularizing or innervating regions or areas of the posterior segment of the eye. In one embodiment, the interior of the eye refers to the posterior segment of the eye, including the posterior chamber, vitreous cavity, choroid, macula, and retina, as well as the blood vessels and nerves vascularizing or innervating regions or areas of the posterior segment of the eye. In this embodiment, intraocular administration refers to administration into the posterior segment of the eye, preferably into the vitreous, and intraocular administration is preferably intravitreal injection.

[0206] In one embodiment, the composition, pharmaceutical composition or medicament of the present invention comprises one or more pharmaceutically acceptable carriers for a formulation suitable for injection.

[0207] In one embodiment, the subject in need of the composition, pharmaceutical composition or medicament of the present invention is administered at least once a day.For example, the composition, pharmaceutical composition or medicament of the present invention can be administered once a day, twice a day or three times a day.In a preferred embodiment, the subject in need of the composition, pharmaceutical composition or medicament of the present invention is administered once a day.

[0208] In another embodiment, the composition, pharmaceutical composition or medicament of the present invention is administered to a subject in need thereof at least once a week. For example, the composition, pharmaceutical composition or medicament of the present invention may be administered once a week, twice a week, three times a week, once a week or up to seven times a week.

[0209] In another embodiment, the compositions, pharmaceutical compositions or medicaments of the invention are administered to a subject in need thereof monthly, twice monthly, every two months, every two or three months, twice yearly or yearly.

[0210] The present invention further relates to a method of treating inflammation in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an agent of the present invention.

[0211] In one embodiment, the method of the present invention is for treating inflammation associated with mononuclear phagocyte accumulation. In a preferred embodiment, the method of the present invention is for treating age-related macular degeneration.

[0212] In one embodiment, the composition, pharmaceutical composition or medicament of the invention is administered to a subject.

[0213] Another object of the present invention is a method of inhibiting CD47 activity in a subject in need thereof, comprising administering to the subject an effective amount of an agent of the present invention.

[0214] Another object of the present invention is a method for eliminating mononuclear phagocyte accumulation in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a prophylactic and / or therapeutic agent as described above.

[0215] Another object of the present invention is a method for removing mononuclear phagocyte accumulation in a subject in need thereof, thereby treating inflammation associated with mononuclear phagocyte accumulation, comprising administering to the subject a therapeutically effective amount of a prophylactic and / or therapeutic agent as described above.

[0216] The present invention also relates to a kit comprising at least one agent, pharmaceutical composition or medicament according to the invention.

[0217] In one embodiment, the kit of the present invention further comprises a means for administering the agent, pharmaceutical composition or medicament to a subject in need thereof.

[0218] In one embodiment, the kit of the invention further comprises instructions for administering the agent, pharmaceutical composition or medicament to said subject.

[0219] In one embodiment, the kit of the present invention is a kit of parts, wherein a first part comprises at least one agent that activates CD47 according to the present invention and a second part comprises at least one other agent that activates CD47 according to the present invention. In a specific embodiment, the kit of the present invention is a kit of parts, wherein a first part comprises at least one agent that directly activates CD47 according to the present invention and at least one agent that indirectly activates CD47 according to the present invention. In a preferred embodiment, the kit of the present invention is a kit of parts, wherein a first part comprises an agent that activates CD47 according to the present invention and an agent that activates Fas according to the present invention.

[0220] In another embodiment, the kit of the invention comprises two parts, a first part comprising at least one agent, pharmaceutical composition or medicament according to the invention, and a second part comprising an additional prophylactic and / or therapeutic agent, which in one embodiment is another agent for treating inflammation, in particular AMD.

[0221] In one embodiment, the components of the kit-of-parts of the invention may be administered separately, sequentially, simultaneously, in parallel or chronologically staggered.

[0222] In one embodiment, the kit of the invention is used (or is intended for use in treating) inflammation.

[0223] In one embodiment, the kit-of-parts part comprising the additional prophylactic and / or therapeutic agent is in a form suitable for the same route of administration as the at least one agent, pharmaceutical composition or medicament of the invention, hi another embodiment, the kit-of-parts part comprising the additional prophylactic and / or therapeutic agent is in a form suitable for a different route of administration than the at least one agent, pharmaceutical composition or medicament of the invention. [Brief explanation of the drawings]

[0224] [Figure 1]A series of histograms showing CD47-mediated mononuclear phagocyte elimination by TSP1. (A) Quantification of subretinal IBA-1+ mononuclear monocytes in 2-3-month-old and 12-month-old C57BL6 / J wild-type, Tsp1- / -, Cd47- / -, and Cd36- / - mice (n = 6-9 per group, one-way ANOVA / Bonferroni test; *p<0.0001 compared to strains in the 2-3-month-old group). (B) Quantification of subretinal IBA-1+ mononuclear monocytes in 2-3-month-old C57BL6 / J wild-type, Tsp1- / -, Cd47- / -, and Cd36- / - mice after 4 days of exposure to 4500 lux of constant green light (n = 6-12 per group, one-way ANOVA / Dunnett test; *p<0.0001 compared to controls). (C) Quantification of subretinal IBA-1+ mononuclear monocytes on the RPE counted at a distance of 0–500 μm to the CD102+ CNV 7 days after laser injury in 3-month-old mice of the indicated strains (n=9–21 / group, Anova / Dunnett, *p<0.0001 compared to control). (D) Quantification of CFSE+ microglial cells of the indicated strains 24 hours after adoptive transfer into C57BL6 / J wild-type mice with or without recombinant TSP1 (10 μg / ml, n=8–16 / group; One-way ANOVA / Bonferroni test, *p<0.0001 compared to C57BL6 / J CSFE+ microglial cells; $p<0.0001 compared to CSFE+ microglial cells of the same strains without TSP1). [Figure 2]A series of graphs showing SNP rs11200638 and HTRA1 expression in leukocytes. (A) Quantitative RT-PCR of Htra1 mRNA normalized to Rps26 mRNA in monocytes isolated from healthy human donor blood after in vitro culture for the indicated times (three preparations yielded similar results). (B) Quantitative RT-PCR of Htra1 mRNA normalized to Rps26 mRNA in lymphocytes, monocytes, and monocytes after 24 hours of culture from healthy human donor blood (three preparations yield similar results). (C) Quantitative RT-PCR of Htra1 mRNA normalized to Rps26 mRNA in lymphocytes and monocytes from fresh blood after 24 h of culture of monocytes from patients with wet AMD homozygous for rs11200638 and age-matched control subjects without the polymorphism (n shown as scatter plot; Mann-Whitney: fresh lymphocytes *p=0.0012; fresh PBMCs *p=0.0353; PBMCs after 24 h *p=0.0312). PBMCs: peripheral blood monocytes. [Figure 3] Figure 1 shows HTRA1-mediated TSP1 degradation. (A) Coomassie staining and Western blot of recombinant TSP1 and recombinant TSP1 co-incubated with recombinant HTRA1 at 37°C. (B) Coomassie staining and Western blot of recombinant TSP2 and recombinant TSP2 co-incubated with recombinant HTRA1 at 37°C. (C) and (D) Quantification of the number of subretinal IBA-1+ mononuclear monocytes on the RPE 7 days after laser injury in 3-month-old wild-type and Tsp1- / - mice that underwent intravitreal injection of CD102+ CNV (n = 9–39 / group; Mann-Whitney *p = 0.0013, PBS-injected Tsp1- / - mice vs. TSP1-injected Tsp1- / - mice; $p = 0.0014, TSP1-injected Tsp1- / - mice vs. rHTRA1-digested rTSP1-injected Tsp1- / - mice). [Figure 4]A series of histograms showing the survival of monocytes (Mo) cocultured with retinal pigment epithelial cells (RPE), a model of subretinal immunosuppression: (i) HTRA-1 counteracts RPE-associated immunosuppression; (ii) simultaneous treatment with the CD47 agonist TSP1 and the FAS agonist MegaFasL rescues immunosuppression after HTRA-1 removal; and (iii) a CD47 agonist peptide rescues HTRA-1-induced immunosuppression in the presence of HTRA-1. (A) Numbers of CFSE+Mo (right panel) and OTX-2+ RPE cells (left panel) after various time points in Mo monoculture (solid line) and Mo / RPE coculture (dotted line) in the presence and absence of HTRA-1. (B) Number of CFSE+ cells after 24 hours of coculture with HTRA-1 followed by 24 hours of control or 24 hours of TSP-1 and MegaFasL (n=3; Anova / Dunnett *p=0.0018). (C) Number of CFSE+ cells after 24 hours of coculture with HTRA-1 and co-stimulation with the control peptide 4NGG or the CD47-stimulating peptide PKT16 (n=3; ANOVA Dunnett *p=0.0286). (D) Number of PU1+ cells after 48 hours of coculture with HTRA-1 and co-stimulation with the indicated concentrations of the control peptide 4NGG or the CD47-stimulating peptides 4NK1 or PKT16 (n=8; ANOVA Dunnett *p<0.0001 compared to control HTRA-1 without peptide). (E) Number of PU1+Mo after 48 hours of co-culture with HTRA-1 and co-stimulation with the indicated concentrations of the control peptide 4NGG or the CD47 stimulatory peptides 4NK1 or the 4N1K-GGGGGGGG-4N1K bipeptide (n=8; ANOVA Dunnett's *p<0.0001 compared to control HTRA-1 without added peptide). [Figure 5]A series of histograms showing the elimination of subretinal mononuclear phagocytes in laser-injured Cx3cr1GFP / GFP mice. Quantification of subretinal IBA-1+ MPs on the RPE counted at a distance of 0–500 μm from the CD102+ CNV 10 days after laser injury in 2-month-old Cx3cr1GFP / GFP mice injected with 2 μl of PBS, recombinant human TSP-1 (10 μg / ml), 4NGG control peptide, or PKHB1 CD47-activating peptide (200 μM) on days 4 and 7 (n=20–25 exposures, Mann-Whitney *p<0.0001). [Figure 6] This series of photographs shows confocal microscopy images of CD11b-CD47 complexes (white dots indicated by arrows) detected by proximity ligation assay on freshly harvested Mφ derived from WT C57BL6 / J (top right) and Cd47- / - mice (bottom right) 1 day after thioglycollate injection. The negative control corresponds to a WT C57BL6 / J mouse without peritonitis induction (left). Hoechst was used for nuclear staining (gray; negative: primary antibody was omitted; experiment was repeated three times with similar results). negCTL = negative control. Scale bar = 10 μm. [Figure 7] A series of histograms showing quantification of CD115+F4 / 80+ICAM-2lo Mo-derived Mφ in exudates from WT C57BL6 / J mice on day 2 after injection with PBS or rTSP-1 (Mann-Whitney p=0.0048); or the control peptide 4NGG or the CD47-activating peptide PKHB1 (Mann-Whitney p=0.0087) on day 1. [Example]

[0225] The present invention will be further understood by reference to the following examples, which are intended to be representative of specific embodiments of the invention and are not intended to limit the scope of the invention.

[0226] material and method Western blot, reverse transcription and real-time polymerase chain reaction and ELISA WB analysis was performed using monoclonal anti-TSP1 antibody (Abcam) as previously described (Houssier et al., PLoS Med. 2008, 5:e39). RT-PCR primers were ordered from Taqman, reference: Hs01016151_m1.

[0227] Microglial cell preparation Microglial cells were prepared from PBS-perfused mice. Brains or retinas were dissociated using the Neural Dissociation Kit Papain (Miltenyi Biotech). The cell suspension was filtered through a 70 μm filter, washed, and resuspended in 75% isotonic Percoll (Percoll Plus, GE Healthcare), then overlaid with 25% Percoll and PBS. The cells were centrifuged at 1000 g for 30 minutes at 4°C. The ring at the interface between the 75% and 25% Percoll fractions was collected, washed with PBS, and centrifuged.

[0228] RPE-Mo co-culture Monocytes In accordance with the Declaration of Helsinki, written and informed consent was obtained from volunteers for studies on human monocyte expression, which were approved by the Ethics Committee of the National Canze-Vingt Hospital Ophthalmology Center (Paris, France) (No. 913572). PBMCs were isolated from heparinized venous blood of healthy subjects by a single centrifugation step on a Ficoll-Paque layer (GE Healthcare) and sorted with EasySep Human Monocyte Enrichment Cocktail without a CD16 Depletion Kit (StemCells Technology). Isolation of mouse peritoneal macrophages, bone marrow-derived monocytes, and photoreceptor outer segments (POS), all in serum-free X-Vivo 15 medium, was performed as previously described (Sennlaub et al., EMBO Mol Med. 2013, 5:1775-1793). For coculture experiments, mononuclear monocytes were stained with CellTrace™ CFSE (Life technologies®) followed by three washes or identified by PU1 immunohistochemistry, which reveals the absence of MP-specific transcription factor expression in the RPE.

[0229] Primary RPE culture Fresh porcine eyeballs were obtained from a slaughterhouse 2–3 hours after enucleation. The eyeballs were cleaned of surrounding tissue and briefly immersed in disinfectant solution (Pursept®). The anterior segment of the eye, including the lens, vitreous body, and vitreous body, was removed. The posterior segment of the eye was washed twice with phosphate-buffered saline (PBS) and then incubated at 37°C in the presence of 0.25% trypsin to detach RPE cells. After 1 hour of incubation, the trypsin solution was removed, and the cells were allowed to recover in Dulbecco's modified Eagle's medium (DMEM) supplemented with inactivated 20% fetal calf serum (FCS) in the presence of antibiotics (1% penicillin / streptomycin). The cells were washed several times and then incubated in a culture dish at 37°C. After 3 days in culture, the cells were again incubated with 0.25% trypsin, washed, and seeded into 48-well culture plates (Grenier®) at a density of 150,000 cells / well (300,000 cells / L or 500 μL / well). After 4 days of incubation at 37°C, the cells reached confluence and optimal cell characteristics (flat cells with good pigmentation) were obtained. To avoid aging of the primary cell cultures, all experiments were performed between days 5 and 7 after seeding into the culture plates.

[0230] Mononcytes-RPE co-culture The RPE cell medium was replaced with FCS-free DMEM (DMEM + 1% penicillin / streptomycin only) the day before co-culture. Monocytes were seeded in 48-well plates at a concentration of 200,000 cells / well in the presence or absence of RPE cells.

[0231] A portion of the co-culture wells was contacted with 1 ng / ml of E. coli-derived lipopolysaccharide (LPS) or recombinant HTRA1 (R&D, 5 μg / ml) to mimic systemic inflammatory activation (LPS) or the microenvironment of AMD (increased HTRA1) (see above). After 24 h of incubation at 37°C, cells were fixed with 4% paraformaldehyde (PAF) for 30 min at 4°C.

[0232] immunohistochemistry After washing the 4% PAF with a solution of PBS-0.1% Triton-0.1% citrate, RPE cells were permeabilized. Nonspecific immunogenic sites were blocked with PBS-0.1% Triton-5% horse serum. After 1 hour, the blocking solution was removed, and the cells were placed in the presence of primary antibodies (polyclonal rabbit anti-human PU.1, 1 / 200, Life Technologies; polyclonal goat anti-human OTX2, 1 / 500, R&D) diluted in PBS-0.1% Triton-1% horse serum for 12 hours at 4°C. After three washes with PBS, secondary antibodies conjugated to fluorescent dyes and diluted in PBS-0.1% Triton-1% horse serum were added along with DAPI (nuclear stain). The cells were incubated at ambient temperature for 1 hour, followed by several washes with PBS.

[0233] Reading and automated quantification by inverted fluorescence microscope (Arrayscan®) Twenty-five fields per well were analyzed using the Arrayscan®, and the number of cells in each culture condition was then counted directly using a computer protocol. Nuclei were all labeled with DAPI, and monocytes were visualized in green at 488 nm (CellTrace™ CFSE) and / or RPE cells in far-red at 647 nm (anti-OTX2 primary antibody recognition). For graphs pooled from quantifications of multiple plates (Figures 4D and 4E), results were expressed as a percentage of the number of PU.1- or OTX2-positive cells normalized to the HTRA1-treated condition.

[0234] Digested TSP1 + liquid chromatography-tandem mass spectrometry (LC-MS / MS) + spectral analysis Trypsin digestion Proteins were reduced by incubation with 5 mM dithiotreitol (AmBic) in 50 mM ammonium bicarbonate for 30 min at 37°C and then alkylated by incubation with 15 mM iodoacetamide in 50 mM AmBic for 30 min at room temperature. Trypsin digestion was carried out overnight in 50 mM AmBic at 37°C with a protein / enzyme ratio of 25 / 1.

[0235] Mass spectrometry analysis Peptide mixtures were analyzed using a U3000 nanoLC (Thermo) coupled to an HCTultra Intrap (Bruker). Formic acid was added to the mixture to a final concentration of 0.1%. Peptides were concentrated and desalted on a precolumn RP-C18 (5 mm, 300 μm id, 100 Å, Thermo) using mobile phase A (2% ACN / 0.1% formic acid) at a flow rate of 20 μL / min for 5 min. They were then separated on an analytical column RP-C18 (15 cm, 75 μm id, 100 Å, Dionex) at a flow rate of 300 nL / min. The elution gradient consisted of 2% to 10% solvent B (95% ACN / 0.1% formic acid) for 10 min, followed by 10% to 35% B for 60 min, and 35% to 50% B for 10 min. The ion trap was operated in positive mode, selecting eight precursor ions from each MS spectrum for fragmentation by collision-induced dissociation (CID). The capillary voltage was set to 2 kV, and full scan spectra were acquired. MSMS spectra were acquired from 100 to 2800 m / z using singly charged ion exclusion, dynamic exclusion for 30 seconds, and an isolation width of 4 Da. The ICC smart target was set to 250,000, and the target mass was set to 622 m / z.

[0236] Tryptic peptides were also analyzed by MALDI-TOF MS in the mass range 700–4000 m / z using α-cyano-4-hydroxycinnamic acid as the matrix (85% CAN, 0.1% TFA, 0.9 mg / mL CHCA in 10 mM ammonium phosphate) on an Autoflex Speed ​​(Bruker) in positive reflectron mode.

[0237] Protein identification For LC-MS / MS data analysis, raw data were processed using Data Analysis 3.4 (Bruker). A signal intensity threshold of 100,000 (AU) and spectral deconvolution were used to generate Mgf files for up to 5,000 compounds. Protein identification was performed using ProteinScape 2.1 (Bruker) using Mascot against the SwissProt database (01 / 04 / 2015) and the Homos sapiens taxonomy (20,203 entries). Trypsin was selected as the enzyme for cleavage residue 2. Cys carbamidomethylation and Met oxidation were set as fixed and variable modifications, respectively; MS tolerance and MS / MS tolerance were set to 0.5 Da. A p-value <0.05 was required for peptide validation. Furthermore, semitrypsin was used as the enzyme and the same parameters were used for analysis.

[0238] PMF analysis of MALDI-TOF data was performed using BioTools 3.2 (Bruker) and Mascot with the following parameters: SwissProt database (01 / 04 / 15), Homos sapiens taxonomy (20,203 entries); trypsin or semitrypsin as enzyme; cleavage residue 1; fixed modification: carbamidomethylation of Cys; variable modification: oxidation of Met; MS tolerance: 60 ppm, and a p-value <0.05 was required for protein validation.

[0239] animal Tsp1 - / - Mouse, CD47 - / - Mouse, CD36 - / - -Mice and Cx3cr1 GFP / GFP Mice were purchased from Charles River Laboratories and Jackson Laboratories. All mice were Crb1 rd8 , Pde6brd1 and Gnat2cpfl3 mutations negative (Tsp1 - / -) or were backcrossed to be negative. Mice were housed in a specific pathogen-free animal facility under a 12 / 12-h light / dark (100-500 lux) cycle and provided with water and regular food ad libitum. All experimental protocols and procedures were approved by the local animal care ethics committee "Comite d'ethique en experimentation animale Charles Darwin" (Nos. Ce5 / 2010 / 011, Ce5 / 2010 / 044, and Ce5 / 2011 / 033).

[0240] Synthesis of PKT16 PKT16 was synthesized using a solid / liquid mixed method. Briefly, 2-chlorotrityl chloride resin was pre-swollen in completely anhydrous CHCl for 2 h. Fmoc-Aa-OH (0.32 mmol) was coupled to 2-CTC resin (400 mg, loading = 1.6 mmol / g) in the presence of diisopropyethylamine (DIPEA, 4 eq.) in CHCl (4 mL). Unreacted sites on the resin were capped by washing with a mixture of CHCl / MeOH / DIPEA (7:2:1) followed by MeOH. After removal of the Fmoc group using 20% ​​piperidine in N,N-dimethylformamide (DMF), chain elongation was carried out with standard Fmoc-protected amino acids (Bachem, Switzerland) using 20% ​​piperidine / DMF for Fmoc deprotection, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole (HBTU / HOBt) for activation, DIPEA as the base, and N-methyl-2-pyrrolidinone (NMP) as the solvent. Upon completion of linear peptide chain assembly, a final MeOH wash (1 × 1 min, 1 × 15 min) was performed to shrink the resin.

[0241] The peptide was cleaved from the resin by two treatments with a HFIP / CHCl cocktail (1:4, v / v) for 15 min each. The reaction mixture was filtered, and the resin was washed sequentially with CHCl and MeOH. The filtrates were pooled, and the solvent was then evaporated under reduced pressure. Finally, the crude linear peptide was precipitated three times with dry-ice-chilled EtO and collected after centrifugation (3 × 5 min, 7800 rpm) and drying (under a nitrogen stream). The crude material was purified by HPLC.

[0242] Laser Damage Model Laser coagulation was performed using a 532 nm ophthalmic laser (Vitra Laser, 532 nm, 450 mW, 50 ms, and 250 μm) attached to a surgical microscope. Two μl of intravitreal injection of HTRA1 and / or TSP1 was performed using a glass capillary (Eppendorf) and a microinjector. The 2 μl injection solution contained 50 μg / ml TSP1 and HTRA1, corresponding to an intraocular concentration of 5 μg / ml, assuming that each protein is diluted approximately 1 / 10 in the intraocular volume. In a series of experiments, we demonstrated that Cx3cr1, which develops excessive subretinal inflammation with age after light stimulation and laser injury, is involved in the development of retinal inflammation. GFP / GFP Mice underwent laser injury (Combadiere et al., J Clin Invest. 2007, 117:2920-2928; Levy et al., EMBO Mol Med. 2015, 7:211-226). On days 4 (when MP infiltration was maximal) and 7 after injury, mice were injected with a 2-μl volume of a 100 μM solution of either PBS, recombinant TSP1 (10 μg / ml), 4NGG control peptide, or the CD47-activating peptide PKT16 (200 μM). On day 10, subretinal inflammation was assessed using flat-mounted RPE / choroidal flat-mounts.

[0243] Light stimulation model As previously described (Sennlaub et al., EMBO Mol Med. 2013, 5:1775-1793), 2- to 3-month-old mice were dark-adapted for 6 h, pupils were dilated, and exposed to green LED light (starting at 2:00 AM, 4500 lux, JP Vezon equipements) for 4 days, then housed under normal facility conditions with a 12-h / 12-h cycle. MP counts were assessed at the end of light exposure or 10 days later (day 14).

[0244] Choroidal and retinal flatmounts for mononuclear phagocyte quantification Eyeballs were enucleated, fixed in 4% PFA for 30 minutes, and incised at the limbus; the cornea and lens were discarded. The retina was carefully peeled off from the RPE / choroid / sclera. The retina and choroid were incubated with anti-IBA-1 (Wako Chemicals) followed by secondary antibody anti-rabbit Alexa 488 (Molecular Probes), followed by Hoechst staining. The choroid and retina were flat-mounted and observed under a DM5500B fluorescence microscope (Leica). IBA-1+ cells were counted in the entire RPE / choroid flat-mount and in the outer segment side of the retina.

[0245] Subretinal adoptive MP transplantation and removal Following Levy et al. (EMBO Mol Med. 2015, 7:211-226), brain microglia from the indicated mouse strains were sorted as described above, labeled with 10 μM CFSE (Life Technologies), washed, and resuspended in PBS. 12,000 cells (4 μL) were injected into the subretinal space of anesthetized 10- to 14-week-old wild-type mice using a glass microcapillary (Eppendorf) and microinjector. A hole was created with the glass capillary prior to subretinal injection to avoid elevated intraocular pressure and allow the retina to detach with 4 μL of solution. Subretinal injection was confirmed by fundus examination. In certain experiments, cells were co-injected with recombinant human TSP1 (10 μg / mL, R&D Systems). 24 hours later, eyes were enucleated, fixed in 4% PFA for 30 minutes, and labeled with DAPI. Eyes exhibiting bleeding were discarded. CFSE+ cells were quantified on the RPE side of the retina and on the apical side of the RPE in the subretinal space in flat mounts.

[0246] Thioglycollate-induced peritonitis and flow cytometry 10-week-old male C57BL / 6J mice and Cd47 - / - Mouse peritoneal exudate cells (PECs) were induced by intraperitoneal injection of 0.5 ml of 3% thioglycollate (T9032, Sigma). One day later, PECs were isolated by flushing the peritoneum with ice-cold PBS. Mφ were negatively selected by magnetic sorting (EasySep Mouse Monocyte Enrichment Kit, Stemcell Technologies) according to the manufacturer's protocol, resuspended in X-VIVO 15 medium (Lonza), and plated onto Lab-Tek® Chamber Slides™ (Nunc®).

[0247] After 2 hours at 37°C under a 5% CO2 atmosphere, cells were rinsed with PBS, fixed with 4% paraformaldehyde for 10 minutes, rinsed, and permeabilized by incubating in 0.1% Triton in PBS for 10 minutes. Duolink® PLA assays were performed according to the manufacturer's instructions (Sigma-Aldrich). Briefly, rabbit anti-CD11b (ab75476, Abcam; 1:1000) and goat anti-CD47 (AF1866, R&D Systems; 1:1000) were incubated overnight at 4°C. Anti-rabbit and anti-mouse oligonucleotide-labeled secondary antibodies (PLA Probes) were then added, followed by signal amplification via ligase and polymerase reactions. Images were captured using an Olympus FLUOVIEW FV1000 confocal laser scanning microscope.

[0248] statistical analysis Graph Pad 6 (GraphPad Software) was used for data analysis and graphical presentation. All values ​​are reported as mean ± SEM. Statistical analysis was performed by one-way ANOVA between means, followed by Bonferroni's post hoc test (for multiple comparisons) or the Mann-Whitney U test (for comparison between two groups), depending on the experimental design. n and P values ​​are indicated in the figure legends.

[0249] Example 1: TSP1 mediates MP clearance via CD47 Tsp1 - / - Mice exhibit increased and prolonged subretinal inflammation after experimentally induced chorioretinitis, light-induced injury, and laser-induced injury, suggesting that TSP1 is involved in the clearance of subretinal mononuclear monocytes (Wang et al., Arch Ophthalmol. 2012, 130:615-620; Ng et al., Invest Ophthalmol Vis Sci. 2009, 50:5472-5478; Chen et al., Am J Pathol. 2012, 180:235-245). The TSP1 receptor mediating this effect has not been identified. Subretinal IBA-1 expression in retinal and RPE / choroid flatmounts from 2-3-month-old and 12-month-old mice was significantly elevated in the subretinal IBA-1 subunits. + Quantification of mononuclear monocytes revealed that Tsp1 - / - Mice and Cd47 - / - There was a significant increase in subretinal mononuclear monocytes with aging in mice, but Cd36 - / - This was not observed in mice (Fig. 1A; all mice had Crb1 rd8 They were backcrossed to eliminate the gene and reared under 100-500 lux at the cage level with no other cage cover and a 12-hour light / dark cycle).

[0250] Similarly, after 4 days of light stimulation, Tsp1 - / - Mice and Cd47 - / - Mice experienced a significant accumulation of subretinal mononuclear monocytes, which continued to accumulate for an additional 10 days after returning to normal light conditions (Fig. 1B; the intensity of our light stimulation model used here was comparable to that of the proinflammatory Cx3cr1GFP / GFP The treatment was adjusted to induce subretinal inflammation in WT mice but not in WT mice (Sennlaub et al., EMBO Mol Med. 2013, 5:1775-1793). Furthermore, 7 days after laser irradiation, Tsp1 - / - Mice and Cd47 - / - In mice, subretinal IBA-1 + There were significantly more mononuclear monocytes (Fig. 1C).

[0251] These results suggest that TSP1 is involved in the removal of subretinal MPs via its receptor CD47.

[0252] Wild-type mice, Tsp1 - / - Mouse or Cd47 - / - In adoptive transplantation experiments, CFSE-labeled brain microglial cells were injected subretinally into wild-type mice. Evaluation of the subretinal microglial population in flat-mounted images 24 hours later revealed that Tsp1 - / - Microglial cells and Cd47 - / - Microglial cells were significantly more resistant to elimination than wild-type microglial cells (Fig. 1D). Co-injection of recombinant TSP1 significantly enhanced the elimination of wild-type microglial cells, and Tsp1 - / - The microglial cell phenotype returned to normal, but Cd47 - / - No effect was observed on microglial cells, confirming that the interaction between TSP1 and CD47 mediates microglial cell elimination (Fig. 1D).

[0253] Taken together, these data suggest that TSP1 eliminates subretinal microglial cells via its CD47 receptor and that this interaction is physiologically important, as both TSP1-deficient and CD47-deficient individuals develop age-, light-, and laser-induced accumulation of subretinal mononuclear phagocytes. - / - Tsp1 is a subretinal mononuclear phagocyte accumulation - / -Since they did not share a common phenotype, our data suggest that this mechanism does not significantly involve CD36 or TGFβ (which is not activated in the absence of CD36).

[0254] Example 2: AMD-associated SNP rs11200638 significantly increases HTRA1 expression in monocyte-derived macrophages Numerous genetic studies have identified chromosome 10q26 as a prime candidate region associated with AMD. The risk haplotype contains SNP rs11200638, which disrupts the CG pattern of a conserved CpG island (DNA methylation site) in the high-temperature-requiring A serine peptidase 1 (HTRA1) promoter (Yang et al., Science. 2006, 314:992-993). This SNP has been shown to remove epigenetic inhibition of HTRA1 transcription in lymphocytes (Yang et al., Science. 2006, 314:992-993). In contrast to macrophages, which can express HTRA1 (Hou et al., Arthritis and rheumatism. 2013, 65:2835-2846), lymphocytes are underrepresented in the retina of AMD patients. To assess HTRA1 expression in monocyte-derived macrophages, we first examined CD14 cells freshly purified from healthy donors and cultured for various lengths of time. +We analyzed HTRA1 expression in peripheral blood monocytes (PBMCs). RT-PCR analysis of HTRA1 revealed rapid and significant induction of HTRA1 during early monocyte-to-macrophage differentiation, which persisted at high levels for at least 168 hours (7 days; Figure 2A). RT-PCR analysis revealed that fresh PBMCs expressed 10-fold more HTRA1 mRNA than fresh blood lymphocytes, and this expression increased another 10-fold after 24 hours of PBMC culture (Figure 2B). Quantitative RT-PCR of patients with wet AMD homozygous for rs11200638 and age-matched control subjects without the polymorphism confirmed that rs11200638 was associated with significantly higher Htra1 mRNA levels not only in lymphocytes but also, more importantly, in PBMCs and early PBMC-derived macrophages, as these cells express and accumulate high amounts of Htra1 in AMD (Figure 2C).

[0255] Taken together, these data confirm that rs11200638 is associated with increased Htra1 transcription in lymphocytes, an observation that extends to mononuclear monocytes, which we have shown to accumulate and play a pathogenic role in AMD.

[0256] Example 3: HTRA1 degrades TSP1 HTRA1 is a relatively nonselective protease and has been shown to degrade multiple proteins (An et al., Invest Ophthalmol Vis Sci. 2010, 51:3379-3386). Interestingly, Coomassie staining of an electrophoresis gel of recombinant HTRA1 (rHTRA1) and recombinant TSP1 (rTSP1) co-incubated at 37°C for 24 hours revealed that HTRA1 degraded TSP1 (Figure 3A). Western blot analysis of the protein confirmed that full-sized TSP1 disappeared under the co-incubation conditions, and multiple smaller bands appeared (Figure 3A). Coomassie staining of an electrophoresis gel of proteins co-incubated with recombinant TSP2 / rHTRA1 did not reveal such degradation (Figure 3B). Next, we analyzed the functionality of rTSP1 and HTRA1-digested TSP1 in laser-induced subretinal inflammation in vivo. Wild-type and Tsp1 mice were intravitreally injected with PBS, rHTRA1, rTSP1, and rHTRA1-digested rTSP1 on day 3. - / - Quantification of laser-induced subretinal inflammation in mice on day 7 revealed that (i) rHTRA1 exacerbated subretinal inflammation in wild-type mice, whereas Tsp1 - / - (ii) rTSP1 significantly reduced this inflammation, but rHTRA1-digested rTSP1 did not (Figure 3C). + Similar differences were observed in associated choroidal neovascularization, measured on day 7 as the surface covered by CNV ( Fig. 3<em>D ).

[0257] Taken together, these results demonstrate that HTRA1 digests TSP1, resulting in a complete loss of TSP1's anti-inflammatory function in laser-induced inflammation in vivo.

[0258] Example 4: HTRA1 cleaves TSP1 between two VVM sites to activate CD47 To characterize the HTRA1 cleavage sites in TSP1, digested fragments of TSP1 were subjected to liquid chromatography-tandem mass spectrometry. This analysis revealed that HTRA1 cleaves TSP1 (i) at a site known to bind to integrin α3β1, (ii) at two sites between the "type 2" domains, and (iii) at two sites between two valine-valine-methionine (VVM) sequences that can interact with the CD47 receptor and are responsible for its efficient CD47 activation. The CD36- or LAP-binding domains of TSP1 were not directly affected.

[0259] These results, along with the observation that TSP1 exerts its immunosuppressive potential through CD47 (Figure 1), suggest that HTRA1 at least partially inactivates CD47, since cleavage by HTRA1 separates the two VVM sites of TSP1. Indeed, the half-maximal effective concentration (EC50) of TSP1 is much lower than that of either the CD47-activating peptide containing only one VVM site or TSP2.

[0260] Example 5: Activation of CD47 reverses the effect of HTRA1 on subretinal immunosuppression in vitro To evaluate the effect of HTRA1 on subretinal immunosuppression, we developed a co-culture model of CFSE-labeled human monocytes and porcine RPE. In this model, CFSE was expressed in the subretinal space, similar to the in vivo adoptive transfer of mononuclear monocytes into the subretinal space (Levy et al., EMBO Mol Med. 2015, 7:211-226). + At least 50% of monocytes are rapidly cleared within 24 hours, but RPE cell numbers (OTX-2 +The number of hMo cells in the coculture (as indicated by nuclear counts) was not affected (Figures 4A and 4B). Addition of recombinant HTRA1 (5 μg / mL) to the cocultures significantly inhibited this RPE immunosuppression, with the number of hMo cells after 24 h being 3-4 times higher than in control conditions (p<0.0001 compared to the coculture control group). Because nuclear RPE markers (see below) were not significantly reduced, the number of RPE cells in the cocultures was automatically counted using OTX2 (Arrayscan). Experiments using transwell plates demonstrated that physical contact between the cells and the RPE cells was required to induce Mo cell death, and that heat inactivation of HTRA1 abolished this effect (not shown).

[0261] To mimic the subretinal microenvironment observed in AMD, cocultures were exposed to recombinant HTRA1 (5 μg / mL). The presence of proteases significantly inhibited RPE immunosuppression (presumably through TSP1 inactivation) and significantly increased monocyte viability (Figure 4A, left panel). HTRA1 did not affect RPE cell viability (Figure 4A, right panel). In an attempt to reverse HTRA1-induced TSP1 inactivation and inhibition of immunosuppression, media and cocultures were treated for an additional 24 hours with either PBS or a mixture of TSP1 (to activate CD47) and MegaFasl (a FAS agonist). After 48 hours, Mo counts in PBS-treated HTRA1-exposed cocultures remained significantly higher compared to the 48-hour control condition. However, when FAS and the CD47 agonist TSP1 were combined, a significant number of Mo were removed, and the number was not significantly different from that of control cultures after 48 hours (Figure 4B). Next, we tested whether the CD47-activating peptide PKT16 could promote Mo removal under HTRA1 coculture conditions. The CD47-activating peptide PKT16 (100 μM) or the 4NGG control peptide was added directly to HTRA1-treated cocultures. CFSE +Monocyte quantification revealed that 4NGG had no significant effect on monocyte numbers, whereas PKT16 completely reversed the HTRA1-induced increase in monocyte viability (Figure 4C, left panel). The number of OTX2+ nuclei remained unchanged, demonstrating no toxicity of 4NGG or PKT16 to RPE cells (Figure 4B, right panel). Furthermore, dose-response experiments revealed that 4N1K and PKT16, but not 4NNG, dose-dependently reduced the number of monocytes (as determined by PU1 immunohistochemistry, which avoids CFSE staining in these experiments) in HTRA1-activated cocultures after 48 h (Figure 4D). Furthermore, HTRA1-activated cocultures were incubated with various concentrations (0 μM, 4 μM, 20 μM, and 50 μM) of the control peptide 4NGG, the CD47-activating peptide 4N1K, or the 4N1K-GGGGGGGG-4N1K peptide (designated d4N1K, Genepep). Quantification after 48 h of culture showed that d4N1K, which binds to two CD47 receptors similar to unhydrolyzed TSP1, was significantly more effective in inducing Mo removal in the coculture model ( Figure 4E ).

[0262] Example 6: Activation of CD47 inhibits laser-induced inflammation-prone Cx3cr1 GFP / GFP Promotes subretinal mononuclear phagocyte clearance in mice in vivo. To evaluate the effects of CD47 activation in vivo, we investigated Cx3cr1, which develops excessive subretinal inflammation with age after light stimulation and laser injury. GFP / GFP Mice underwent laser injury (Combadiere et al., JCI 2007; Levy et al., EMBO Mol Med 2015). On days 4 (when MP infiltration was maximal) and 7 after injury, 2 μl of PBS, TSP1, the control peptide 4NGG, or the CD47-activating peptide PKT16 (100 μM) was injected. Subretinal inflammation was assessed on day 10 using flat-mounted RPE / choroidal flat-mounts.

[0263] The results showed that injection of TSP1 or PKT16 reduced the subretinal IBA-1 observed in the RPE adjacent to the laser irradiation by 10 days after laser irradiation. + It is shown that MPs were removed significantly more efficiently than with PBS or the control peptide 4NGG (FIG. 5).

[0264] Example 7: CD47 activation promotes recMφ elimination during peritonitis To test whether CD47 influences inflammation resolution in other pathological situations, we used a model of acute thioglycollate-induced peritonitis characterized by an early neutrophil accumulation followed by recruited monocyte-derived inflammatory macrophages (recMφ), both of which undergo proapoptotic clearance with different kinetics (Gautier et al., Blood. 2013, 122:2714-2722).

[0265] Proximity ligation assays revealed numerous specific CD11b / CD47 complexes in WT recMφ collected 1 day after peritonitis induction (Figure 6, top right, white dots indicated by arrows). - / - This complex is not observed in mice (Fig. 6, bottom right).

[0266] The experiments also show that a single intraperitoneal injection of recombinant TSP1 or the CD47-specific activating peptide PKHB1 on day 1 significantly promoted the elimination of recMφ, as observed on day 2 (Fig. 7 ).

[0267] These results demonstrate that the CD11b-CD47 complex is present on peritoneal recMφ and that CD47 activation promotes recMφ elimination during peritonitis.

Claims

1. A multimeric peptide comprising at least two peptide monomers linked via a linker, the at least two peptide monomers activate CD47, and the peptide monomers consist of 4N1K (sequence KRFYVVMWKK (SEQ ID NO: 1)); The linker is a Gly-rich linker. Multimeric peptides.

2. The multimeric peptide of claim 1, which is a dimer.

3. The multimeric peptide of claim 1, wherein the multimeric peptide consists of two 4N1K peptides linked via a Gly-rich linker.

4. The multimeric peptide according to any one of claims 1 to 2, comprising or consisting of SEQ ID NO:7.

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