Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the present invention refers to a peptide, multimeric derivatives thereof, pharmaceutical composition thereof and their use in therapy, in particular for inhibiting Tumor necrosis factor-alpha and/or lymphotoxin activity.
• Tumor necrosis factor-alpha (TNF-a, hTNF-a, TNF-alpha or hTNF-alpha) [ACCESSION NUMBER NCBI CAA26669.1 (SEQ ID NO:2)] is a potent pro -inflammatory cytokine exerting pleiotropic effects on various cell types.
• TNF-a elicits a wide variety of responses, including fever, synthesis of acute-phase proteins, increased vascular permeability, T and B- cell activation and migration thereof, cell proliferation and apoptosis [1-3].
• TNF-a is generated in precursor form, known as trans-membrane TNF-a, expressed as a cell surface type II polypeptide on activated macrophages, lymphocytes and other cell types [4, 5].
• TNF-a- converting enzyme TNF-a- converting enzyme
• Soluble TNF-a is a homotrimer of 17-kDa cleaved monomers and transmembrane TNF-a also can exist as a homotrimer of 26-kDa uncleaved monomers [12].
• TNF-a is involved in the acute inflammatory response to stimuli, such as infection or tissue injury, and also plays a critical role in the pathogenesis of chronic inflammation and chronic inflammatory diseases, such as rheumatoid arthritis (RA) and Crohn's disease [13].
• RA rheumatoid arthritis
• TNF-a also plays a central role in the pathogenesis of psoriasis, psoriatic arthritis and ankylosing spondylitis [6].
• TNF-a has been proposed as a therapeutic target for a number of diseases.
• Five anti-TNF-a agents (Infliximab, Etanercept, Adalimumab, Cetolizumabpegol and Golimumab) have been successfully introduced for the treatment of chronic inflammatory diseases. All anti-TNF-a agents are effective against RA, though with very different efficacies, and not all of them are effective against Crohn's disease [14]. Thus the need for new anti-TNF drugs is still pressing.
• an anti-TNF-a peptide was selected from a phage library. It was synthetized as a tetra-branched molecule and has been in vitro characterized for inhibition of TNF-a binding to its receptors, which is the first property for an anti-TNF-a agent to be considered a potential drug for anti-inflammatory therapies.
• the peptide molecule synthetized in a monomeric or branched form, is able to bind to the pro inflammatory human factor TNF-alpha.
• the peptide of the invention is also able to bind to human lymphotoxin (LT) [accession number NCBI BAA00064.1 (SEQ ID NO: 5)], a protein involved in inflammation and acting on the same TNF-a receptors.
• LT lymphotoxin
• the peptide of the invention inhibits the binding of TNF-alpha and/or LT to its receptors present on cells and block the onset of inflammatory process triggered by TNF-alpha and/or
• the peptide of the invention presents the properties of an anti TNF-alpha agent and represents a potential drug for anti- inflammatory therapies.
• HIHDDLLRYYGW SEQ ID NO: 1
• the peptide is in a linear or multimeric form.
• the peptide in the linear form is produced through synthetic or recombinant route, and the peptide in the multimeric form is produced through synthetic route.
• MAP Multiple Antigenic Peptide
• R is a peptide as above defined;
• X is an amino acid having at least two functional amino groups.
• X is lysine, ornithine, nor-lysine or amino-alanine.
• the peptide of the invention is for use as anti-TNF-alpha agent and/or anti- lymphotoxin (LT) agent.
• LT lymphotoxin
• the peptide of the invention is for use in the treatment and/or prevention of a pathology characterized by overproduction of TNF-alpha and/or LT.
• the peptide of the invention is for use in the treatment and/or prevention of an inflammatory pathology.
• the inflammatory pathology is acute or chronic, more preferably the inflammatory pathology is selected from the group consisting of: rheumatoid arthritis,
• a further object of the invention is a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a peptide as above defined.
• the pharmaceutical composition is for systemic, oral or topical administration.
• a further object of the invention is a method of treatment and/or prevention of a pathology characterized by overproduction of TNF-alpha and/or LT comprising administering in a subject in need thereof an effective amount of the peptide as above defined.
• a pathology is an inflammatory pathology.
• the inflammatory pathology is acute or chronic.
• the inflammatory pathology is selected from the group consisting of: rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriasis and psoriatic arthritis, atopic dermatitis, lupus, juvenile idiopathic arthritis, cystic fibrosis.
• a further object of the invention is a functional fragment of the peptide of SEQ ID NO: 1.
• the fragment is a peptide having a percentage of identity of at least 45 %, preferably at least 75 %, more preferably at least 85 %, even more preferably at least 90 % with SEQ ID NO: 1.
• the functional fragment is able to inhibit the binding of TNF-alpha and/or LT to its receptors.
• Said fragment is a peptide of at least 5 amino acids, preferably at least 8 amino acids, more preferably at least 10 amino acids.
• Preferably said fragment is for use as a medicament, more preferably for use as anti-Tumor necrosis factor (TNF)-alpha agent and/or anti-lymphotoxin (LT) agent, even more preferably for use in the treatment and/or prevention of a pathology characterized by overproduction of TNF-alpha and/or LT.
• TNF tumor necrosis factor
• LT anti-lymphotoxin
• the fragment is for use in the treatment and/or prevention of an inflammatory pathology, more preferably the inflammatory pathology being acute or chronic, even more preferably the inflammatory pathology is selected from the group consisting of: rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriasis and psoriatic arthritis, atopic dermatitis, lupus, juvenile idiopathic arthritis, cystic fibrosis.
• an inflammatory pathology more preferably the inflammatory pathology being acute or chronic, even more preferably the inflammatory pathology is selected from the group consisting of: rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriasis and psoriatic arthritis, atopic dermatitis, lupus, juvenile idiopathic arthritis, cystic fibrosis.
• the invention relates also to a polynucleotide coding for the peptide comprising the amino acid sequence from the amino to the carboxyl terminus: HIHDDLLRYYGW (SEQ ID NO : 1), preferably for medical use.
• the polynucleotide is for use as anti-Tumor necrosis factor (TNF)-alpha agent and/or anti-lymphotoxin (LT) agent, more preferably for use in the treatment and/or prevention of a pathology characterized by overproduction of TNF-alpha and/or LT. Even more preferably, the polynucleotide is for use in the treatment and/or prevention of an inflammatory pathology.
• TNF tumor necrosis factor
• LT anti-lymphotoxin
• the inflammatory pathology is acute or chronic, even more preferably the inflammatory pathology is selected from the group consisting of: rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriasis and psoriatic arthritis, atopic dermatitis, lupus, juvenile idiopathic arthritis, cystic fibrosis.
• the invention relates also to a vector comprising the above polynucleotide.
• Said vector is preferably for medical use.
• the vector is for use as anti-Tumor necrosis factor (TNF)-alpha agent and/or anti-lymphotoxin (LT) agent, more preferably for use in the treatment and/or prevention of a pathology characterized by overproduction of TNF-alpha and/or LT.
• TNF tumor necrosis factor
• LT anti-lymphotoxin
• the vector is for use in the treatment and/or prevention of an inflammatory pathology.
• the inflammatory pathology is acute or chronic, even more preferably the inflammatory pathology is selected from the group consisting of: rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriasis and psoriatic arthritis, atopic dermatitis, lupus, juvenile idiopathic arthritis, cystic fibrosis.
• the invention relates also to a host cell genetically engineered which expresses the peptide as above defined.
• said host cell is for medical use.
• the host cell is for use as anti-Tumor necrosis factor (TNF)-alpha agent and/or anti-lymphotoxin (LT) agent, more preferably for use in the treatment and/or prevention of a pathology characterized by overproduction of TNF-alpha and/or LT.
• TNF tumor necrosis factor
• LT anti-lymphotoxin
• the host cell is for use in the treatment and/or prevention of an inflammatory pathology.
• the inflammatory pathology is acute or chronic, even more preferably the inflammatory pathology is selected from the group consisting of: rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriasis and psoriatic arthritis, atopic dermatitis, lupus, juvenile idiopathic arthritis, cystic fibrosis.
• the polynucleotide is selected from the group consisting of: R A or DNA, preferably said polynucleotide is DNA.
• the vector is an expression vector selected from the group consisting of: plasmids, viral particles and phages.
• said host cell is selected from the group consisting of: bacterial cell, fungal cell, insect cell, animal cell and plant cell, preferably said host cell is an animal cell.
• the peptides of the invention are in the form of synthetic or recombinant, linear and multimeric peptides in any chemical, physical and/or biological form such as to have an anti TNF-alpha and/or LT activity.
• the peptides of the invention can be produced according to the methods known by the experts in the art, e.g. by chemical synthesis or by recombination.
• the peptides of the invention are synthetized and used in linear form, soluble and multimeric, even bound to substrates, as e.g. on a polyacrylamide skeleton of dextran units or of ethylene glycol units.
• the peptides of the invention are synthetized and used in the branched form as Multiple Antigenic Peptide (MAP), as disclosed e.g. in the patent US 5,229,490.
• MAP Multiple Antigenic Peptide
• compositions can be chosen on the basis of the treatment requirements.
• Such compositions are prepared by blending and are suitably adapted to systemic, oral or topical administration, and as such can be administered in the form of tablets, capsules, oral preparations, powders, granules, pills, injectable, or infusible liquid solutions, suspensions, suppositories, preparation for inhalation.
• Tablets and capsules for oral administration are normally presented in unit dose form and contain conventional excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g. polyvinylpyrrolidone and starch derivatives such as sodium glycolate starch), coloring agents, flavoring agents, and wetting agents (for example sodium lauryl sulfate).
• excipients such as binders, fillers (including cellulose, mannitol, lactose), diluents, tableting agents, lubricants (including magnesium stearate), detergents, disintegrants (e.g. polyvinylpyrrolidone and starch derivatives such as sodium glycolate starch), coloring agents, flavoring agents, and wetting agents (for example sodium lauryl sulfate).
• the oral solid compositions can be prepared by conventional methods of blending, filling or tableting.
• the blending operation can be repeated to distribute the active principle throughout compositions containing large quantities of fillers. Such operations are conventional.
• Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or with a suitable vehicle before use.
• Such liquid preparations can contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel, or hydrogenated edible fats; emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which can include edible oils), such as almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired, conventional flavoring or coloring agents.
• Oral formulations also include conventional slow-release formulations such as enterically coated tablets or granules.
• composition for administration by inhalation can be delivered from an insufflator or a nebulizer pressurized pack.
• parenteral administration fluid unit dosages can be prepared, containing the compound and a sterile vehicle.
• the compound can be either suspended or dissolved, depending on the vehicle and concentration.
• the parenteral solutions are normally prepared by dissolving the compound in a vehicle, sterilising by filtration, filling suitable vials and sealing.
• adjuvants such as local anaesthetics, preservatives and buffering agents can also be dissolved in the vehicle.
• the composition can be frozen after having filled the vials and removed the water under vacuum.
• Parenteral suspensions are prepared in substantially the same manner, except that the compound can be suspended in the vehicle instead of being dissolved, and sterilized by exposure to ethylene oxide before suspending in the sterile vehicle.
• a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the compound of the invention.
• compositions may be tablets, lozenges, pastilles, or gel.
• the compounds can be pharmaceutically formulated as suppositories or retention enemas, e.g. containing conventional suppositories bases such as cocoa butter, polyethylene glycol, or other glycerides, for a rectal administration.
• Topical formulations can contain for example ointments, creams, lotions, gels, solutions, pastes and/or can contain liposomes, micelles and/or microspheres.
• ointments examples include oleaginous ointments such as vegetable oils, animal fats, semisolid hydrocarbons, emulsifiable ointments such as hydroxystearin sulfate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water soluble ointments containing polyethylene glycols of various molecular weights.
• Creams as known to formulation experts, are viscous liquids or semisolid emulsions, and contain an oil phase, an emulsifier and an aqueous phase.
• the oil phase generally contains petrolatum and an alcohol such as cetyl or stearic alcohol.
• Formulations suitable for topical administration to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
• transdermal delivery comprises conventional aqueous and non-aqueous vectors, such as creams, oils, lotions or pastes or can be in the form of membranes or medicated patches.
• the compounds of the present invention may be employed alone as a sole therapy or in combination with other therapeutic agents for the prevention and/or treatment of the above- mentioned conditions.
• the combination can be administered as separate compositions (simultaneous, sequential) of the individual components of the treatment or as a single dosage form containing both agents.
• the active ingredients may be separately formulated into single-ingredient preparations of one of the above-described forms and then provided as combined preparations, which are given at the same time or different times, or may be formulated together into a two- or more- ingredient preparation.
• the peptides as above defined as above defined may be administered to a patient in a total daily dose of, for example, from 0.1 to 50 mg/kg body weight daily. Dosage unit compositions may contain such amounts of submultiples thereof to make up the daily dose. The determination of optimum dosages for a particular patient is well known to one skilled in the art. As is common practice, the compositions are normally accompanied by written or printed instructions for use in the treatment in question.
• FIG. 1 Structure of anti-TNF-a peptides synthesized in tetra-branched form. Amino acids of the four peptide sequences are indicated as one letter code.
• TNF-a from 250 nM to 2 ⁇ , was injected over biotin-coupled linear (A) or tetra-branched (B) peptide immobilized on a SA sensor chip.
• A biotin-coupled linear
• B tetra-branched
• C biotin-coupled tetra-branched peptide
• Adalimumab was captured via Protein A on a CM5 sensor chip as described in Methods. hTNF-a was flowed in the presence of different concentrations (1, 10 and 25 ⁇ ) of branched anti-TNF peptide.
• B Curve of binding inhibition of hTNF-a binding to Adalimumab derived by experiment in A. y axis indicates the percentage of binding between Adalimumab and hTNF-a; x axis represents the concentration of tetra-branched anti-TNF peptide. The IC50 is indicated.
• A Biacore sensorgrams illustrating binding of 3 nM hTNF-a to immobilized TNFR2, in the presence of different concentrations of branched peptide, from 10 ⁇ to 1 ⁇ .
• B Curve of binding inhibition of hTNF-a to TNFR2 derived by experiment in A.
• y axis indicates the percentage of binding between TNFR2 and TNF-a;
• x axis represents the concentration of tetra-branched anti-TNF peptide.
• the IC 50 is indicated.
• Figure 5 The branched anti- hTNF-a peptide inhibits TNF-a binding to human melanoma cells.
• the red signal is due to hTNF-a binding (3 nM) to membrane receptors present on A375 cells (A).
• Incubation of hTNF-a with the branched anti-hTNF- ⁇ peptide (1, 10, 20 ⁇ ) produces a dose-dependent decrease in the red signal (B-D). Nuclei were stained with DAPI (blue).
• the high intensity of the peak of monomeric TNF-a was the result of homo-trimer dissociation caused by laser energy generated during sample desorption.
• Random 12-mer peptide phage library Ph.D.12TM (New England Bio labs) was incubated with biotinylated hTNF-a. 10 11 phage, diluted in PBS (137 mM NaCl, 2 mM KC1, 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , pH 7.4) containing 0.1% (v/v) Tween 20 (PBST) and 3% bovine albumin (BSA) and incubated with 50 nM biotinylated hTNF-a o/n at 4°C, was added to 100 ⁇ Dynabeads-M280 coupled with Streptavidin (Invitrogen), saturated with 5% BSA in PBST, for 15 minutes at room temperature.
• PBS 137 mM NaCl, 2 mM KC1, 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , pH 7.4
• BSA bovine albumin
• Solid-phase synthesis was carried out on a MultiSynTech Syro automated multiple peptide synthesizer (Witten, Germany), employing Fmoc chemistry with 2-(lH-benzotriazole-l-yl)- 1 , 1 ,3,3-tetramethyluronium hexafluoro-phosphate/N,N-diisopropylethylamine (HBTU) activation.
• HBTU hexafluoro-phosphate/N,N-diisopropylethylamine
• Branched peptides were synthesized on Fmoc-4-Lys2-Lys Tentagel resin.
• Fmoc-Lys-(Biotin)-OH (Iris Biochem GmbH) was used as first group coupled to the solid phase and Fmoc-PEG-OH (Iris Biochem GmbH) as second.
• Peptides were cleaved from the resins and deprotected by treatment with trifluoroacetic acid containing water and triisopropylsilane (95:2.5:2.5). After precipitation with diethyl ether, all peptides were purified by HPLC using a CI 8 Jupiter column (Phenomenex) and characterized by UltraflexIII MALDI TOF/TOF mass spectrometry (Bruker).
• Biotinylated linear and tetra-branched TNF-peptides were immobilized on a SA sensor chip. Briefly, peptides diluted in HBS-EP+ (10 mM Hepes, 157 mM NaCl, 2,4 mM EDTA , p20 0,05 % pH 7,4) were injected for 60 sec at a flow rate of 10 ⁇ /minute, obtaining 300 and 700 RUs respectively. Different concentrations of hTNF-a, from 250 nM to 2 ⁇ in HBS- EP+, were injected for 180 sec at a flow rate of 75 ⁇ /min onto immobilized peptides.
• Human TNFR2 fused to an antibody FC fragment (TNFR Human, Prospec), diluted in HBS- EP+ at a concentration of 10 ⁇ g/ml, was injected for 120 sec at a flow rate of 5 ⁇ /min on a CM5 sensor chip previously coated with Protein A (6500 RUs).
• Competition experiments were carried out injecting 3 nM hTNF-a for 180 sec at flow rate of 20 ⁇ /min with various concentrations (from 1 ⁇ to 10 ⁇ ) of tetra-branched anti-hTNF-peptide. Regeneration was achieved with a 30-second pulse of glycine 10 mM pH 2.2.
• A375 human melanoma cells were grown in DMEM, supplemented with 10% fetal bovine serum, 200 mg/mL glutamine, 100 mg/mL streptomycin, and 60 mg/mL penicillin. Cells were maintained at 37°C in 5% C0 2 .
• A375 cells were plated at a density of 3x10 4 per well in 24 well plates with cover glass slides. Samples were fixed through incubation with a PBS-4% paraformaldehyde (PFA) solution for 15 min, saturated for 30 min at 37°C with PBS-1% BSA and incubated with 500 nM biotinylated hTNF-a and different concentration (20, 10 and 1 ⁇ ) of anti-TNF-a peptide for 30 min at room temperature, then with 0.5 ⁇ g/ml Streptavidin-Atto 550 (Sigma- Aldrich) for 15 min at room temperature. Nuclei were stained with DAPI.
• PFA paraformaldehyde
• hTNF-a binding to receptors was analyzed by confocal laser microscope (Leica TCS SP5) with 364/555 nm excitation and 458/570 nm emission filters for DAPI and Atto 550, respectively. All images were processed using Image J software (NIH).
• the anti-TNF- ⁇ peptide was incubated in a 3-fold or 10-fold molar excess with respect with hTNF-a, at room temperature for 5 min.
• the dissociation of hTNF-a was analysed by mass spectrometry using a sinapinic acid matrix (Sigma-Aldrich) and a protein standard II (mass range -10,000-70,000 Da, Brucker Daltonics) in an ultrafieXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) in linear mode.
• a commercial phage library (Ph.D.12, NEB) was used to identify 12-mer peptides that bind human TNF-a (hTNF-a). Three cycles of biopanning were carried out against soluble biotinylated hTNF-a. Elution of specific anti-TNF-a peptides was achieved by addition of Adalimumab monoclonal antibody. A parallel biopanning was carried out by surface plasmon resonance (SPR) using Biacore T100.
• SPR surface plasmon resonance
• phage library was injected over biotinylated hTNF-a, previously immobilized on a streptavidin (SA) sensor chip, and bound phages were eluted by competition with Adalimumab monoclonal antibody.
• SA streptavidin
• the phage ELISA performed to detect TNF-a-specific peptides, selected by conventional biopanning or BiacoreTlOO phage selection, revealed several positive clones, which DNA analysis showed to express the same amino acid sequence: HIHDDLLRYYGW (SEQ ID NO: l).
• the selected peptide was synthesized in a tetrameric form (Figure 1) through standard Fmoc solid phase synthesis, building the tetramer on a three-lysine core [15].
• the tetra-branched peptide structure was chosen because it strongly increases peptide stability to circulating proteases, thus improving half- life with respect to corresponding monomeric sequences [16- 18].
• the branched structure constructed with short peptides is generally non immunogenic [19]. Stability and low immunogenicity are of great importance for possible use in vivo [20- 25].
• both the tetra-branched peptide and the corresponding monomeric sequence were also synthesized with a C-terminal biotin.
• hTNF-a was injected over linear and tetra-branched peptides previously immobilized on a Biacore T100 SA sensor chip. hTNF-a was flowed at concentrations ranging from 250 nM to 2 ⁇ . To assess any non-specific binding, hTNF-a was also injected over an empty flow cell. The linear ( Figure 2A) and tetra-branched peptides ( Figure 2B) bound hTNF-a in a dose-dependent manner. The peptides proved specific for hTNF-a since they did not show any binding for murine TNF-a (not shown).
• TNF-a-specific peptide synthesized in linear and tetrameric form showed practically the same kinetics (KD 3x10 ⁇ 6 M and 6x10 ⁇ 6 M, respectively).
• Human lymphotoxin (LT) a member of the TNF superfamily, has 34% sequence identity with hTNF-a. Together with TNF-a, it is implicated in inflammation and autoimmunity and is also a target of an existing anti- inflammatory drug [26].
• LT Human lymphotoxin
• inventors In the attempt to verify possible cross reaction of the anti-TNF-a peptide with TNF-a and LT, inventors also analyzed its binding for LT. Surprisingly, binding affinity proved stronger for LT (KD 1.2xlO _11 M) than for TNF-a, as shown in Figure 2C.
• Branched anti-TNF- ⁇ peptide inhibited binding of TNF-q to cells expressing TNF-q receptors
• FIG. 6A shows MS analysis of hTNF- a alone, indicating the peaks corresponding to monomeric, dimeric and trimeric forms.
• Figure 6B shows MS analysis of hTNF-a after incubation for 5 minutes with a threefold molar excess of the peptide with respect to TNF-a. The peak corresponding to the homo- trimeric form disappeared. With a tenfold molar excess of peptide, the homo-dimeric form also disappeared ( Figure 6C).
• TNF-a is an attractive target for the development of anti-inflammatory drugs because it is an important mediator in the pathogenesis of several inflammatory diseases, including RA, Crohn's disease and ankylosing spondylitis.
• Very severe symptoms are already treated with anti-TNF-a recombinant antibodies or recombinant receptors, that act by sequestering the soluble TNF-a and preventing its binding to receptors.
• Five anti-TNF-a agents have been successfully introduced into clinical practice for chronic inflammatory diseases [14] but they have some major side effects [28-31]. Since they are also very expensive it is important to identify new anti-TNF- ⁇ agents to enlarge the panel of possible therapeutic agents for inflammatory diseases.
• the peptide identified by phage selection efficiently competed with the antibody Adalimumab and significantly also with natural receptors of TNF-a. This inhibition was demonstrated at molecular level by Biacore ( Figure 4) and directly on viable cells expressing TNF-a receptors ( Figure 5). The latter result is important because it shows that the peptide actually inhibits the onset of inflammation-boosting reactions triggered by the binding of TNF-a on cell receptors. Interestingly, we found that the peptide bound strongly to LT, another protein involved in inflammation and acting on the same TNF-a receptors. LT is already known to be a target of Etanercept but not of Adalimumab or Infliximab [33].
• This invention leads to the identification and optimization of a new anti TNF-alpha active ingredient for development of a new drug for human or animal use.
• TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell 53, 45-53.

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