WO2014083232A1 - Use of peptides or antibodies against n-procalcitonin for the treatment of pulmonary lesions - Google Patents

Abstract

The invention relates to the use of an isolated peptide, an antibody or a fusion protein capable of binding to N-PCT, and of the compositions and pharmaceutical forms that include same, in the production of a drug for the treatment of pulmonary lesions.

Description

 Use of anti-N-procalcitonin peptides or antibodies for the treatment of lung lesions.

The present invention is within the field of biotechnology and medicine. It refers to the use of peptides and / or antibodies capable of binding to N-procalcitonin in the preparation of a medicament with anti-inflammatory activity for the treatment of pulmonary lesions, and more specifically, indirect pulmonary lesions, caused for example by Response Syndrome Systemic inflammatory (SIRS), sepsis, severe sepsis and septic shock, trauma, pancreatitis, multiple transfusions of blood products, and also in particular, for the treatment of acute lung lesions and septic shock.

STATE OF THE PREVIOUS TECHNIQUE The inflammatory response that causes dysfunction and failure of an organ remains the main problem after an injury in many clinical conditions, such as sepsis. Under these conditions, multiple organ dysfunction syndrome (MODS) causes organ failure. Acute lung injury (ALI) that manifests clinically with an acute respiratory distress syndrome (ARDS) is a major component of the MODS of various etiologies.

ALI and ARDS are acute respiratory failure syndromes characterized by an intense pulmonary inflammatory response, which includes neutrophilic presence, interstitial edema, rupture of epithelial integrity, and parenchymal lung injury (Ware & Matthay 2000. N Engl J Med; 342: 1334-49).

Although many modalities of therapy for ALI have been tested, such as with glucocorticoids (Thompson 2003. Crit CareMed 31: 253-257), monoclonal anti-endotoxin antibodies (Manocha et al., 2002. Expert Opin Investig Drugs 1 1: 1795-1812) , therapy with antitumor necrosis factor alpha (TNF-a) [Leeper-Woodford et al., 1991. Am Rev Respir Dis 143: 1076-1082] and interleukin (IL) -1 therapy, the results have been disappointing. It has been suggested that clinical outcomes in patients with ALI / ARDS would improve with pulmonary inflammation modulation (Brower 2001., Chest 120: 1347-1367]. Inflammatory mediators play a key role in the pathogenesis of ARDS, which is the leading cause of death in these conditions.

In experimental animal models of sepsis and clinical sepsis, a high number of bacterial compounds initiate excessive production and release of cytokines and chemokines. The characteristics of these chemokines and cytokines, as well as the high concentrations of family members of the CT (calcitonin) peptides obtained from the CALC-I gene, including PCT (procalcitonin) and N-PCT (aminoprocalcitonin) (Becker 2004. J. Clin. Endocrine !. Metab. 89, 1512-1525) also correlate with the severity of sepsis (Boveris et al., 2002. Free Radie. Biol. Med. 33, 1 186-1193; Joshi et al., 2003 FEBS Lett. 555, 180-184 .; Victor et al., 2004. Int. Immunopharmacol. 4, 327-347).

Procalcitonin (PCT or proCT) is a glycopeptide prohormone of 1,116 amino acids and approximately 13kDa molecular weight. This molecule is the precursor of calcitonin. Its synthesis begins with the transcription of the CALC-I gene located on chromosome 1 1 p. Subsequently, this transcript is processed giving rise to preprocalcitonin, precursor of PCT. This precursor is composed of 141 amino acids and subsequent processing leads to PCT. Its amino acid sequence was already described in 1984 (Moullec et al. 1984. FEBS lett. 167: 93-97)

This PCT for its part undergoes successive digestions to give rise to 3 different molecules: aminoprocalcitonin (N-procalcitonin, N-PCT or N-proCT) composed of 57 amino acids from the N-terminal zone; immature and non-active calcitonin, consisting of 33 amino acids from the central zone of the PCT; and peptide corresponding to the C-terminal zone, formed by 21 amino acids (residues 96-1 16 of the PCT) and called CCP-I or katacalcin (Jacobs et al. 1981. J Biol Chem. 256: 1803-2807; Steenbergh et al. 1986. FEBS lett. 209: 97-103). Under normal (non-pathological) physiological conditions, these molecules are produced as a result of a proteolytic intracellular process carried out by the prohormone convertase enzyme in the thyroid C cells and in the neuroendocrine cells of the lung.

N-PCT was first described in rats as a 57 amino acid neuroendocrine peptide derived from the N-terminal half of the PCT (Burns et al., 1989. Mol. Endocrino !. 3, 140-147). The synthesis of N-PCT is complex and begins with the translation of a 141-amino acid pre-prohormone [PPCT (pre-procalcitonin)] and the formation of its immediate PCT precursor, a peptide of 1,16 amino acids (Le Moullec et al., 1984. FEBS Lett. 167, 93-97; Steenbergh et al., 1986. FEBS Lett. 209, 97-103).

During acute systemic inflammation, particularly related to a bacterial infection, the specific control of Calc-1 tissues is broken and there is an increase in the expression of the CALC-I gene that causes a release of PCT and N-PCT from all tissues parenchymatous and differentiated types of cells throughout the body, without a corresponding secretion of CT (Müller et al., 2001. J. Clin. Endocrine !. Metab. 86,396-404), and, more importantly, their levels persist over relatively long periods of time and correlate with the severity and mortality of sepsis [Assicot 1993. Lancet 341, 515-518 .; Whang et al., 1998. J. Clin. Endocrinol Metab 83, 3296-3301).

Specific immunoneutralization of N-PCT can inhibit lethality induced by LPS in rats. The ability of anti-N-PCT to inhibit the production, release or action of proinflammatory cytokines is partially responsible for the protective effect of anti-N-PCT. Our research suggests that N-PCT, in addition to being a reliable marker of severity and mortality, plays a crucial role in septic shock and directly affects the outcome by regulating the production of crucial pro and anti-inflammatory factors for development. of this syndrome N-PCT can act as an important mediator for neuroimmune actions and could serve as a circulatory afferent signal for the activation of inflammatory responses to sepsis. However, there is no information regarding the effects of N-PCT on ALI.

The nuclear factor (NF) -kB is a fundamental transcription factor that is vital for the expression of genes related to inflammation, such as NOS and inflammatory cytokines. NF-κΒ is normally retained in the cytoplasm, where it is bound by an inhibitory protein known as ΙκΒ. Stimulation of ΙκΒ kinases leads to phosphorylation, ubiquitization and degradation of ΙκΒα which allows NF-κΒ to be translated into the nucleus and induce transcription. Previous studies indicate that most of the agents that activate NF-κΒ transmit their effects by suppressing ilaciónκΒα IKB phosphorylation and subsequent degradation. Nuclear factor (NF) -kB has been known as a main route for the development of ALI during sepsis (Fan et al., 2001. Am J Physiol Lung Cell Mol Physiol 281: L1037-L1050). However, it remains unknown if anti-N-PCT has protective effects on severe ALI induced by sepsis and, if applicable, if NF-kB inhibition plays a role in them.

DESCRIPTION OF THE INVENTION

The authors of the present invention have shown that peptides capable of binding to N-PCT / PCT, or anti-N-PCT / PCT are capable of restoring lung levels of CALC-I mRNA and procalcitonin, reducing lung lesions, decreasing proinflammatory cytokines, while at the same time increasing the expression of anti-inflammatory cytokines, inhibiting the activation of NF-κΒ, and improving survival in sepsis. The peptides, antibodies or fragments thereof capable of binding to the free N-PCT as well as the N-PCT that is part of the procalcitonin molecule (PCT, composed of N-procalcitonin, calcitonin and katacalcin or C-terminal) will have the technical effect claimed in the present invention.

Thus, a first aspect of the invention relates to the use of an isolated peptide, hereinafter peptide of the invention, or an antibody, or a fragment thereof, hereinafter antibody or antibody fragment of the invention, with the ability to bind N-PCT / PCT, in the preparation of a medication for the treatment of lung lesions in mammals.

The term "isolated," as used herein, refers to nucleotides or peptides that: 1) are substantially free of components that normally accompany or interact with it in nature, or 2) if they are found in their natural environment, they have been synthetically (not naturally) altered by human intervention and / or introduced into a cell that does not possess them natively. For example, a natural nucleotide becomes "isolated" if it has been altered, or if it comes from a DNA that has been altered through human intervention (through, for example, but not limited to, directed mutagenesis, insertions, deletions, etc.) In the same way, a natural nucleotide becomes "isolated" if it is introduced by unnatural means into a genome not native to said nucleotide (transfection). Therefore, the term "isolated" in the latter case is equivalent to the term "heterologous."

The term "peptide", as used herein, refers to a polymer formed by the binding, in a defined order, of alpha-amino acids by a peptide bond, and includes modifications or derivatives thereof, for example, glycosylation, phosphorylation. , acetylation, amidation, etc.

The amino acids of the peptide of the invention, depending on the orientation of the amino group carrying the alpha carbon atom, can belong to the L series or the D series, preferably, to the L series. Additionally, the amino acids can be natural amino acids or modified or uncommon amino acids. Among the natural amino acids are aliphatic amino acids (glycine, alanine, valine, leucine and isoleucine), hydroxylated amino acids (serine and threonine), sulfur amino acids (cysteine and methionine), dicarboxylic amino acids and their amides (aspartic acid, asparagine, glutamic acid and glutamine), the amino acids that have two basic groups (Usina, arginine and histidine), aromatic amino acids (phenylalanine, tyrosine and tryptophan) and cyclic amino acids (proline). Illustrative, non-limiting examples of modified or uncommon amino acids include 2- aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4- aminobutyric acid, 6-aminocaproic acid, 2- aminoheptanoic acid, 2- aminoisobutyric acid, 3-aminoisobutyric acid, 2- aminopimelic acid, 2,4-diaminobutyric acid, desmosin, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, alo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alo-isoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, orinithine, etc.

N-procalcitonin or N-PCT is understood herein as a 57 amino acid neuroendocrine peptide derived from the N-terminal half of the PCT. The synthesis of N-PCT is complex and begins with the translation of a 141-amino acid pre-prohormone [PPCT (pre-procalcitonin)] and the formation of its immediate PCT precursor, a 116 amino acid peptide. It is therefore a peptide of sequence SEQ ID NO: 1. Therefore, in a preferred embodiment of this aspect of the invention, the N-PCT is a sequence peptide SEQ ID NO. one

 (APFRSALESSPADPATLSEDEARLLLAALVQDYVQNKASELEQEQEREGSSLDSPRS) or, alternatively, SEQ ID NO: 2

(PFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRS).

In this specification, a variant or a biologically active fragment of the sequences SEQ ID NO: 1 or SEQ ID NO: 2 is also considered as N-PCT. The isolated peptide or the antibody or the fragment thereof of the invention are characterized by their N-PCT / PCT binding capacity, and, advantageously, for its ability to inhibit the biological activity of N-PCT / PCT. The binding capacity of a peptide or the antibody or fragment thereof to the N-PCT / PCT can be determined by any appropriate method that allows determining the binding between two molecules (eg, by an affinity test), said method comprising contacting the N-PCT / PCT with the peptide to be tested under conditions that allow the binding of said peptide to the N-PCT / PCT and evaluate the binding between the peptide and the N-PCT / PCT. In a particular embodiment, said affinity test can be performed, for example but not limited to, using the Surface Plasmon Resonance (SPR) technique, or similar techniques using radiolabeled N-PCT / PCT, or, alternatively, radioactively marking The peptide to be tested. In general, this type of affinity assay comprises contacting the N-PCT, eg, immobilized in the wells of a plate, with the peptide whose binding capacity to N-PCT is desired to know, and, after incubating for a period of appropriate time, analyze the binding of the peptide to the N-PCT / PCT. Peptides with low affinity for N-PCT / PCT are removed by washing while peptides with higher affinity remain bound to N-PCT / PCT and can be released by breaking the molecular interactions between both molecules, which can be done , for example, lowering the pH.

Advantageously, the peptide and / or the antibody of the invention are characterized not only by their ability to bind N-PCT / PCT, but also by their ability to inhibit the biological activity of N-PCT / PCT, and , consequently, indirectly, regulate or temporarily or temporarily inhibit the activity of NF-κΒ. Although it is not desired to be linked to any theory, it is believed that the ability of a peptide or of An antibody to inhibit the biological activity of N-PCT / PCT is due to the direct binding of said peptide or antibody to N-PCT / PCT. The ability of a peptide or an antibody to inhibit the biological activity of N-PCT can be analyzed, in vitro, by any appropriate conventional method illustrative of such effect, eg:

 a) by an assay based on the measurement of cell proliferation in a culture of effector T lymphocytes, in the presence of an anti-CD3 antibody, Treg lymphocytes and tritiated thymidine, and in the presence or absence of the peptide to be tested; O well

 b) by an assay based on the co-culture of splenocytes of transgenic OT-I mice (mice in which T lymphocytes have a T-cell receptor specific for the SIINFEKL peptide (SEQ ID NO: 4) of the ovalbumin) with Treg lymphocytes in the presence of antigen [SIINFEKL peptide (SEQ ID NO: 4)], in the presence or absence of Treg lymphocytes, and in the presence or absence of the peptide to be tested; or alternatively c) by an assay based on a mixed lymphocyte response (MLR) in which effector cells of a mouse (eg, BALB / c) are mixed with dendritic cells obtained from another strain of mouse (eg, C57BL / 6 ) in the presence or absence of Treg lymphocytes obtained from a mouse of one of the strains (eg BALB / c) and in the presence or absence of the peptide to be tested. Similarly, similar experiments can be performed using Treg lymphocytes of human origin. The peptide or antibody of the invention, capable of binding to the N-PCT can be identified by different techniques. Thus, for example, but not limited to, the technology associated with phage libraries called Biopanning developed by Smith, G.P., (1985), Science 228: 1315 can be used. This technique allows to identify peptides that have a high affinity binding with a specific protein (in this case, N-PCT / PCT), and then quantify, by means of in vitro tests, the ability of the different peptides to inhibit the biological activity of said protein The sequence of the peptides that bind to the N-PCT / PCT can be deduced from the corresponding DNA sequence after several "biopanning" cycles (generally 3).

Various variations of the biopanning technique presented by Smith have been described and reference is made to Christian ef a /., (1992) J. Mol. Biol., 227: 711; Cwiria et al., (1990) Proc. Nati Acad. Sci. USA, 87: 6378; Culi et al., (1992) Proc. Nati Acad. Sci. USA, 89: 1865; Huís et al., (1996) Nature Biotechnol., 7: 276; and Bartoli et al., 1998 Nature Biotechnol., 16: 1068, W098 / 54312, U.S. Patent No. 5,582,981, Balass et ai, (1996) Anal. Biochem., 243: 264, the SELEX method, US Patent No. 5,475,096, WO99 / 06542A

Therefore, in a preferred embodiment, the peptide of the invention is obtained by a method comprising:

 a) incubating a library of phage-exposed peptides is incubated with a peptide of sequence SEQ ID NO: 1, or a biologically active variant or fragment thereof,

 b) let the phage-exposed peptides bind with the sequence peptide SEQ ID NO: 1 or a biologically active variant or fragment thereof (this step is called panning), use binding interactions so that only the peptides Specific ones presented by the bacteriophage bind to the target. For example, but not limited to, the selection of an antibody presented by the bacteriophage with antigen coated on microtiter plates.

 c) Washing stage: unbound phages are removed. Only phages united with strong affinity remain.

 d) The specifically bound phages are eluted in acidic medium. The eluted pool of phages is amplified in vivo and the procedure is repeated.

In a preferred embodiment of this aspect of the invention, the variant of SEQ ID NO: 1 is SEQ I D NO: 2, and the fragment of SEQ I D NO: 1 is SEQ I D NO. 3 (QEREGSSLDS PRS).

The end result is that the peptides produced by the bacteriophage are specific. After several cycles, the individual clones are isolated and sequenced.

In another preferred embodiment, the antibody of the invention is obtained by a method, hereinafter the first method of generating antibodies of the invention, comprising:

 a) immunize a mammalian animal with a peptide according to (c), b) extract the antiserum from the animal, and optionally

 c) purify the antibody that specifically recognizes N-procalcitonin.

In a more preferred embodiment, the method further comprises adding a cysteine at one end of a peptide of the invention before step (a). Yet more preferably, it comprises conjugating the peptide with KLH (Keyhole Limpet Hemocyanin).

Therefore, in another more preferred embodiment, the antibody of the invention is obtained by a method comprising:

 a) adding a cysteine at one end of a peptide of sequence SEQ ID NO: 1 or to a biologically active variant or fragment thereof,

 b) conjugate the peptide with KLH (Keyhole Limpet Hemocyanin), c) immunize a mammalian animal with a peptide according to (c), d) extract the antiserum from the animal, and optionally

 e) purify the antibody that specifically recognizes N-procalcitonin.

Another aspect of the present invention relates to a method for generating antibodies (hereafter second method of generating antibodies of the invention) comprising the following steps:

 a) immunizing a mammalian animal with a peptide of sequence SEQ ID NO: 1, of sequence SEQ ID NO: 2, or a biologically active variant or fragment thereof,

 b) analyze the titre against the peptide of step (a) by an immunological technique, in the mammalian animal of step (a),

 c) fusing the splenocytes of host animals for the generation of immortalized cell lines,

 d) expand the clones,

 e) select the best producers.

In a preferred embodiment, the second antibody generation method of the invention further comprises adding a cysteine at one end of a peptide of sequence SEQ ID NO: 1, sequence SEQ ID NO: 2, or a variant or fragment biologically active thereof, before immunization of the animal. Even more preferably, it comprises conjugating it with KLH (Keyhole Limpet Hemocyanin).

Therefore, in an even more preferred embodiment, the second antibody generation method of the invention comprises: a) adding a cysteine at one end of a peptide of sequence SEQ ID NO: 1 or to a biologically active variant or fragment thereof,

 b) conjugate it with KLH (Keyhole Limpet Hemocyanin),

 c) immunize a mammalian animal with a peptide according to (g), d) analyze the titre against the peptide of step (g) by ELISA, in the mammalian animal of step (h),

 e) fusing the splenocytes of host animals for the generation of immortalized cell lines,

 f) expand the clones,

 g) select the best producers.

 In another preferred embodiment, the peptide of step (a) of the first or second method of the invention is the sequence peptide SEQ ID NO: 3. SEQ ID NO: 3 corresponds to the amino acid sequence of the last 13 residues of the N-PCT protein.

Additionally, the first or second method of the invention may include a previous step, which consists in the generation of the peptide or the peptides of the invention, from step (a), in a recombinant manner. Therefore, in another preferred embodiment, the antibody of the invention has been obtained by the first or second method of the invention, wherein the peptide of step (a) is a recombinant peptide.

In this report "animal" means any organism of the Eukaryota superreino and Metazoa kingdom. The term "mammal" is used to refer to any organism of the Eukaryota super kingdom, Metazoa kingdom, Chordata phylum, Craniata subphylum, Gnathostomata superclass and Mammalia class.

Another aspect of the invention relates to a fusion protein comprising:

 a) a peptide and / or an antibody of the invention; Y

 b) a transporter peptide capable of internalizing a peptide in a cell.

A "transport peptide capable of internalizing a peptide in a cell", sometimes identified in this description as a "transport peptide", is a peptide capable of crossing the cell membrane and penetrating a cell from the outside, a characteristic that can be conferred to the peptide (v. g., peptide of the invention) to which it is fused (fusion protein of the invention) thereby providing an alternative to the transport of peptides of interest (eg, peptides of the invention) into the target cells. This mechanism of entry of peptides into the cell is known as "protein transduction or delivery". Various transport peptides with the ability to internalize a peptide in a cell are known (Schwarze SR et al., Science, 1999 Sep 3; 285 (5433): 1569-72; Niesner U. et al., Bioconjug. Chem. 2002 Jul- Aug; 13 (4): 729-36; Ford KG et al., Gene Therapy, 2001; 8: 1-4; and Gusarova GA et al., J. Clin. Invest. 2007 Jan; 117 (1): 99 -1 11). Virtually any transporter peptide capable of internalizing a peptide in a cell can be used for the implementation of the present invention; however, in a particular embodiment, said carrier peptide is a peptide comprising a segment "PTD" (from the English "protein transduction domain"). Illustrative, non-limiting examples of proteins comprising said protein transduction domains (PTD) include the TAT protein (transacting translational protein) of the human immunodeficiency virus 1 (HIV-I), the homeotic transcription factor ( Antp) of Drosophila antennapedia and the VP22 DNA binding protein of simple herpesviruses 1 (HSV-I), although it has also been suggested that this property of internalizing peptides in cells is possessed by other proteins such as influenza virus hemaglutinin, lactoferrin , fibroblast growth factor 1, fibroblast growth factor 2 and Hoxa-5, Hoxb-4 and Hoxc-8 proteins (Ford KG et al., Gene Therapy, 2001; 8: 1-4).

In a particular embodiment, said carrier peptide is a peptide derived from the HIV-I TAT protein, comprising the sequence responsible for the transduction of peptides, whose basic domain (PTD) comprises residues 49-57 of said HIV TAT protein -I, specifically, the amino acid sequence RKKRRQRRR (SEQ ID NO: 5), or residues 47-57 of said HIV-I TAT protein, such as the peptide whose amino acid sequence is YGRKKRRQRRR (SEQ ID NO: 6) or the peptide whose amino acid sequence is CGISYGRKKRRQRRR (SEQ ID NO: 7). In another particular embodiment, said carrier peptide is a peptide derived from the D. antennapedia Antp protein, which comprises the antennapedia homeodomain (AntpHD) comprising the domain responsible for transduction of peptides (PTD) [residues 43-58 of d icha Antp protein), which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 8), or a functional fragment of the same. In another particular embodiment, said carrier peptide is a peptide derived from HSV-I VP22 protein that comprises a domain responsible for peptide transduction (PTD). In another particular embodiment, said carrier peptide is a peptide derived from the ARF tumor suppressor protein (alternative reading frame) comprising the amino acid sequence responsible for the ability of the peptide to penetrate cells, such as the fragment that it comprises residues 26-44 of said ARF protein, namely, the KFVRSRR PRT ASCALAFVN aminoacid sequence (SEQ ID NO: 9), or a fragment thereof comprising residues 37-44 of said ARF protein, namely, the amino acid sequence SCALAFVN (SEQ ID NO: 10). The peptide of the invention may be attached to any of the terminal (amino or carboxyl) ends of the transporter peptide capable of internalizing a peptide of the invention in a cell. Thus, in a particular embodiment, the carboxyl terminal end of the peptide of the invention is attached to the amino terminal end of said carrier peptide, while, in another particular embodiment, the amino terminal end of the peptide of the invention is attached to the end. carboxyl terminal of said carrier peptide. The peptide of the invention can be directly or not directly linked to said transport peptide capable of internalizing a peptide in a cell. Therefore, in a particular embodiment, the peptide of the invention [peptide (i)] is directly linked to said carrier peptide [peptide (ii)], while in another particular embodiment, the peptide of the invention [peptide ( i)] is linked to said transporter peptide [peptide (ii)] through a spacer peptide ("Hnker" or "spacer") between said peptides (i) and (ii). Accordingly, if desired, the fusion protein of the invention may further contain a spacer peptide located between said peptide of the invention [peptide (i)] and said carrier peptide [peptide (ii)]. Advantageously, said spacer peptide is a structurally flexible peptide, such as a peptide that gives rise to an unstructured domain. Virtually any peptide with structural flexibility can be used as a spacer peptide; however, illustrative, non-limiting examples of such spacer peptides include peptides containing amino acid residue repeats, eg, Gly and / or Ser, or any other suitable repetition of amino acid residues. Optionally, if desired, the fusion protein of the invention may include a amino acid sequence useful for the isolation or purification of the fusion protein of the invention. Said sequence will be located in a region of the fusion protein of the invention that does not adversely affect the functionality of the peptide of the invention. Virtually any amino acid sequence that can be used to isolate or purify a fusion protein (generically referred to as "tag" or "tag" peptides) may be present in said fusion protein of the invention. By way of illustration, not limitation, said amino acid sequence useful for isolating or purifying a fusion protein can be, for example, an arginine tail (Arg-tag), a histidine tail (His-tag), FLAG-tag, Strep -tag, an epitope capable of being recognized by an antibody, such as c-myc-tag, SBP-tag, S-tag, calmodulin binding peptide, cellulose binding domain, chitin binding domain, glutathione S- transferase-tag, maltose binding protein, NusA, TrxA, DsbA, Avi-tag, etc. (Terpe K., Appl. Microbiol. Biotechnol. (2003), 60: 523-525), β-galactosidase, VSV-glycoprotein (YTDIEMNRLGK) (SEQ ID NO: 1 1), or an amino acid sequence such as: Ala His Gly His Arg Pro (SEQ ID NO: 12) (2, 4, and 8 copies), Pro lie His Asp His Asp His Pro His Leu Val He His Ser (SEQ ID NO: 13), etc.

Therefore, in a preferred embodiment of this aspect of the invention, the carrier peptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. More preferably, the fusion protein further comprises an amino acid sequence useful for the isolation or purification of the fusion protein of the invention. Another aspect of the invention relates to a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising a therapeutically effective amount of a peptide and / or an antibody of the invention, or a fusion protein of the invention, together with at least one pharmaceutically acceptable excipient.

In a preferred embodiment, the pharmaceutical composition of the invention further comprises another active ingredient. In an even more preferred embodiment of the invention, the other active ingredient is a compound that inhibits or regulates the activity of N F-KB, preferably with anti-inflammatory activity. The compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art. Thus, they can be, without limitation, in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals. Alternatively, the compositions may be prepared for administration in a solid manner or any other type of administration.

Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising a peptide and / or an antibody of the invention, a fusion protein of the invention, or a pharmaceutical composition of the invention. .

In this specification, "pharmaceutical form" means the mixture of one or more active ingredients with or without additives that have physical characteristics for proper dosage, preservation, administration and bioavailability.

Another aspect of the invention relates to the use of a peptide and / or an antibody of the invention, a fusion protein of the invention, a pharmaceutical composition of the invention, or a pharmaceutical form of the invention, in the preparation of a medicament for the treatment of lung lesions, or alternatively a peptide and / or an antibody of the invention, a fusion protein of the invention, a pharmaceutical composition of the invention, or a pharmaceutical form of the invention, for its use in the treatment of lung lesions. In a preferred embodiment of this aspect of the invention, the lung injury is an indirect lung injury. In another preferred embodiment, the lung injury is an acute lung injury.

In this report it is understood as "indirect lung injury", which is due to the release into the bloodstream of substances such as cytokines or the like, which activate complex mechanisms of aggregation of neutrophils, capillary endothelial injury and activation of coagulation, which systemically injure different organs, of which one of the most vulnerable is the lung.

This complex mechanism of molecule activation causes a deterioration of lung function, with intrapulmonary short circuit and hypoxemia. The main feature of this form of presentation is a diffuse, patchy and bilateral lung injury.

The most typical examples are sepsis of extrapulmonary origin that exerts an authentic multiplier effect and is usually present in almost all cases, being considered a fundamental factor in the gestation of the syndrome and situations in which the treatment of shock required large transfusions of blood products and plasma expander solutions. Another aspect of the invention relates to the use of a peptide and / or an antibody of the invention, a fusion protein of the invention, a pharmaceutical composition of the invention, or a pharmaceutical form of the invention, in the Preparation of a medicament for the treatment of acute respiratory distress syndrome, or alternatively a peptide and / or an antibody of the invention, a fusion protein of the invention, a pharmaceutical composition of the invention, or a pharmaceutical form of the invention , for use in the treatment of acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome or adult respiratory distress syndrome (ARDS). It was described by Ashbaugh and Petty in 1967 (Lancet 2 (751 1): pp. 319-23) and its current definition was established in 1994 by the American-European Consensus Conference: bilateral pulmonary infiltrates of acute onset, in the absence of congestion pulmonary (PCP <18 mmHg, measured by pulmonary artery catheter) and a Pa02 / Fi02 <200 ratio. During the years that have passed since then, this definition has received different criticisms, most of which are due to the use of the Pa02 / Fi02 relationship without reference to the values of inspiratory and expiratory pressures with which it has been measured. Using a consensus process, a panel of experts, gathered at the initiative of the ESICM, the ATS and the SCCM, developed during 2011 a definition that tries to be viable, reliable and valid and whose applicability could be objectively evaluated.

A provisional definition with three exclusive severity categories based on the degree of hypoxia was proposed using a minimum PEEP level of 5 cm H20:

 1) Medium: Pa02 / Fi02≤ 300;

 2) Moderate: Pa02 / Fi02≤ 200 and

 3) Serious: Pa02 / Fi02 ≤ 100. (The notion of acute lung injury is eliminated), and with four auxiliary variables for severity: degree of radiographic alteration, respiratory system compliance ≤ 40 mL / cm H20, PEEP ≥ 10 cm H20 and corrected exhaled minute volume ≥ 10 L / m. The time span from the insult or the worsening of the lungs to the appearance of the symptoms became longer (one week) was also modified; guidelines for radiographic interpretation were established and the origin of pulmonary edema was defined as not fully explained by cardiac failure or water overload, emphasizing the need to objectively assess it by echocardiography in the absence of risk factors.

This definition was evaluated in a large retrospective cohort from previous clinical trials. The auxiliary variables did not show predictive value in terms of prognosis, so they were deleted from the definition. The resulting definition proved to have a higher predictive value for mortality than that of the previous definition. This was for the different stages of 27% (95% CI 24% -30%); 32% (95% CI 29% -34%); and 45% (95% CI 42% -48%), respectively; P <0.001.

Another aspect of the invention relates to the use of a peptide and / or an antibody of the invention, a fusion protein of the invention, a pharmaceutical composition of the invention, or a pharmaceutical form of the invention, in the Preparation of a medicament for the treatment of systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis and septic shock. Alternatively, it refers to a peptide and / or an antibody of the invention, a fusion protein of the invention, a composition Pharmaceutical of the invention, or a pharmaceutical form of the invention, for use in the treatment of SIRS, sepsis, severe sepsis and septic shock. The concept of sepsis includes from the host SRIS to the suspected or documented serious infection. Severe sepsis is characterized either by acute alteration of the function of one or more organs (hemodynamic, renal, respiratory, hepatic, hematological or neurological function), or poor tissue perfusion (hyperlactacidemia) or arterial hypotension (transient or persistent) .

Septic shock is defined by the presence of arterial hypotension that does not respond to intravascular volume expansion and requires amines infusion for treatment.

Definitions of sepsis

Systemic inflammatory response syndrome (SRIS): It is considered to be present when there are two or more of the following four clinical findings:

1. Body temperature above 38 ° C or below 36 ° C.

 2. Heart rate greater than 90 beats per minute.

 3. Hyperventilation, evidenced by a respiratory rate greater than 20 per minute or a PaC02 less than 32 mm Hg.

 4. WBC count greater than 12,000 or less than

4,000 ^ L cells or with 10% immature forms.

Sepsis: any documented or suspected infection with one

or more of the following criteria:

 • Fever (central temperature> 38.3 ° C) or hypothermia (temperature

central <36 ° C).

 • Tachycardia> 90 beats / minute.

 • Tachypnea> 30 breaths / minute.

 • Alteration of consciousness.

 • Edema or positive balance> 20 ml / kg in 24 h.

Hyperglycemia (plasma glucose> 1 10 mg / dL) in the absence of

diabetes.

 • Leukocytosis (> 12,000 / mm3) or leukopenia (<4,000 / mm3) or count

normal with> 10% immature forms.

 • High plasma levels of C-reactive protein or procalcitonin.

Svc02> 70% or cardiac index> 3.5 l / min / m2. Severe sepsis: episode of sepsis associated with organic dysfunction, hypoperfusion or hypotension attributable to sepsis.

 • Hypoxemia with Pa02 / FI02 <300 mmHg.

 • Oliguria (diuresis <0.5 ml / kg / h for at least 2 hours).

Creatinine increase> 0.5 mg / dl or value> 2 mg / dl.

 • Coagulation disorder (INR> 1, 5 or TTPa> 60 secs).

 • Thrombocytopenia <100,000 / mm3.

 • Hyperbilirubinemia (bilirubin> 2.0 mg / dl).

 • Hyperlactacidemia (> 3 mmol / l or 24 mg / dl).

· Arterial hypotension (TAS <90 mmHg, TAM <70 or decrease in

the TAS> 40 mmHg).

Septic shock: persistent arterial hypotension that cannot be

explained by causes other than sepsis, and that does not recover

despite resuscitation with adequate volume.

 INR: International Normalized Radio; Sv02: oxygen saturation

of hemoglobin in central venous blood; TAS: blood pressure

systolic; TAM: mean blood pressure; TTPa: thromboplastin time

partial activated.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention it refers to the peptide (s) of the invention, the antibody (s) of the invention, a fusion protein of the invention, a pharmaceutical composition of the invention, or a pharmaceutical form of the invention, or to a co mposition, or a pharmaceutical form or preparation that includes them.

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different diagnostic effect, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a planned modified manner that provides the specific activity or effect.

The present invention means biologically active variant or fragment, those variants or fragments of the indicated peptides that have the same physiological, metabolic or immunological effect, or have the same utility as those described. That is, they are functionally equivalent. Such effects can be determined by conventional methods such as those described in the examples that accompany this description. In particular, the term "variant" refers to a peptide substantially homologous to any of the peptides whose amino acid sequence is recorded in SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3. In general , a variant includes additions, deletions or substitutions of amino acids. The term "variant" also includes peptides resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation.

As used herein, a peptide is "substantially homologous" to any of the peptides SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3, when its amino acid sequence exhibits a good alignment with the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3, respectively; that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3, respectively, of at least 50%, typically of at least 80%, advantageously of at least 85%, preferably of at least 90%, more preferably of at least 95%, and even more preferably of at least 99% The sequences homologous to any of the peptides SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3, can be easily identified by a person skilled in the art, for example, with the help of a computer program appropriate to compare sequences. The term "identity", as used herein, refers to the proportion of identical nucleotides or amino acids between two nucleotide or amino acid sequences that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the GAG program, including GAP (Devereux et al., Nucleic Acids Research 12: 287 (1984) Genetics Computer Group University of Wisconsin, Madison, (Wl); BLAST, BLASTP or BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 (1999).

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Alterations in the pulmonary expression levels of the CALC-I gene and procalcitonin in animals with simulated operation and septic animals treated with vehicle or Anti-N-PCT 20 hours after ligation and cecal puncture (CLP) . Data are presented as mean ± SE (n = 10): ** P □ 0.001 versus the mock group † P □ 0.05 versus the vehicle group.

Fig. 2. Alterations in lung water content in simulation animals and septic animals treated with vehicle or Anti-N-PCT 20 hours after ligation and cecal puncture (CLP). Data are presented as mean ± SE (n = 10): ** P □ 0.001 versus the mock group † P □ 0.05 versus the vehicle group. Fig. 3. Morphological alterations of the lungs determined by photomicrography.

A) Photomicrograph of a lung section of a simulated operated rat.

B) Photomicrograph of a lung section of a septic rat 22 hours after ligation and cecal puncture (CLP) treated with vehicle. C) Photomicrograph of a lung section of a septic rat 20 hours after CLP treated with anti-NPCT. D) Histopathological score of lung lesion in animals with simulated operation and of septic animals treated with vehicle or anti-N PCT 20 hours after CLP. Data are presented as means ± SE (n = 5) ÷ * P □ 0.05 versus simulated group; + P □ 0.05 versus vehicle group. Fig. 4. Pulmonary activity of myeloperoxidase (MPO). CLP resulted in increased lung MPO activity compared to the group of simulated operation animals. The induced MPO was reduced at 20 h after anti-NPCT treatment. * p □ 0.001 compared to simulated treatment, + p □ 0.01 compared to LPC + anti-NPCT.

Fig. 5. Alterations in bacterial load in peritoneal fluid A), blood B), and lungs C) in the group of animals with simulated operation and septic animals treated with vehicle or anti-NPCT 20 hours after ligation cecal and puncture (CLP). Data were presented as means ± SE (n = 10): * p □ group 0.01 vs. your vehicle.

Fig. 6. Alterations in lung levels of tumor necrosis factor (TNF) -a (A), IL-6 (B) ΙΙ-1β (C) 11-10 (D) MIP-2 (E) Rantes (F) in animals with simulated operation and septic animals treated with vehicle or anti-NPCT 20 hours after ligation and cecal puncture (CLP). Data are presented as mean ± SE (n = 10): * P □ 0.0 versus simulated group, + P □ 0.0 versus vehicle group.

Fig. 7. Alterations in nuclear levels of nuclear factor (NF) -kB p65 A), cytoplasmic levels of IkBa B) in the lungs of animals with simulated operation and septic animals treated with vehicle or anti-NPCT 20 hours after ligation and cecal puncture (CLP). Data are expressed as means ± SE (n = 10) ÷ * P □ 0.05 versus animals with simulated operation; # P □ 0.05 against animals with LPC treated with vehicle.

Fig. 8. Alterations in the survival rate 96 hours after ligation and cecal puncture (CLP) with the vehicle and with anti-CLP N PCT treatment. There were 22 animals in each group. The survival rate was estimated by Kaplan-Meier. EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

MATERIALS AND METHODS USED

Animal model of sepsis

Adult male Wistar rats (270-325 g) (Animal Production and Research Center, University of Seville, Seville, Spain) were used in a temperature controlled enclosure with a light cycle: 12-hour darkness and fed with a diet of nutritionally balanced rodent (2014S; Harían Ibérica). Before the induction of sepsis, the rats fasted overnight but were allowed to drink water ad libitum. Sepsis was induced by ligation and cecal puncture (CLP). After anesthesia with a perotineal injection of ketamine (100 mg / kg) and xylazine (5 mg / kg), a 2 cm incision was made in the abdominal wall, and the blind was carefully extruded. Approximately 25% of the cecum was ligated just below the ileocecal valve to prevent bowel obstruction. The cecum was punctured twice using a sterile 16 gauge needle and squeezed to expel fecal material into the peritoneal cavity. The muscle and skin layers of the abdomen were closed. All previous manipulations were carried out by the same surgeon to ensure consistency. This stretch of linked blind was chosen after the evaluation of multiple ligation distances of cecal sections. This distance was chosen because it resulted in approximately 80% mortality in control animals. Immediately after the procedure, the animals were given 20 mL / kg of saline to allow recovery. In a group of animals with simulated operation (n = 6) the abdomen was open and the blind man manipulated, but no cecal ligation or cecal puncture was performed, and the abdomen was closed. All experiments were carried out in accordance with the guidelines on animal use and care established by the Council of the Directive of the European Communities (86/609 / EEC) and Spanish legislation (BOE / 67: 8509/1988) , and were approved by the Institutional Committees of Ethics and Research of the University Hospital of Valme. Anti-N-PCT Administration

The rabbit polyclonal anti-humanCT precursor (AdB Serotec (1720-9670) was dissolved in 1% PBS / BSA to a final concentration of 100 μΜ. The endotoxin levels of the anti-N-PCT preparations were measured by the method Limulus amoebocyte lysate, as previously described (Yang et al., 2001. Biochim Biophys Acta 1537: 167-174) The results showed that the levels of endotoxin in the preparations were undetectable.An anti-N solution was boosted. PCT or a vehicle 3 hours before implantation by means of two hundred microliter Alzet mini-pumps (infusion rate, 8 μΙ / h) Rats that suffered CLP were treated well with anti-N-PCT (200 μg / Kg, intraperitoneally ) or with a vehicle two hours after the CLP, followed by a continuous infusion of anti-N-PCT (1200 g / Kg) or a vehicle by means of a minipump for 20 hours. Twenty hours after the CLP (ie , 20 hours after the implantation of the minipump), the ra These were sacrificed and blood and tissue samples were taken. The total dose of anti-N-PCT that each rat received was 1200 μg / kg body weight.

Determination of water content and histological examination Prior to the induction of systemic inflammation, each animal was anesthetized as in Example 1. Pulmonary edema was estimated by comparing wet to dry weight ratios. The lung tissues were dried in an oven at 70 ° C for 48 hours. The water content in the lungs was measured as described previously (Wu et al., 2007. Am J Respir Crit Care Med Vol 176. pp 805-813). Morphological alterations in the lungs were examined 20 hours after the onset of sepsis by means of an optical microscope.

For the histopathological evaluation, the freshly collected lung was fixed in a 10% formalin phosphate buffer, encapsulated in sucrose, frozen in dry ice using an OCT compound, and cryo-sectioned. Sections were stained with hematoxylin / eosin using standard techniques.

Morphological examinations were carried out using an optical microscope and documented with photographs. An evaluation system was used to estimate the degree of lung damage based on the following histological characteristics: edema, hyperemia and congestion, neutrophil marginalization and infiltration of tissues, foreign bodies and intraalveolar hemorrhage, and cellular hyperplasia. Each characteristic was evaluated as absent, mild, moderate or severe, with a score of 0-3. A total score for each animal was calculated (Bachofen & Weibel 1982. Clin Chest Med 3: 35-56).

Measurement of myeloperoxidase activity in lung tissue.

Neutrophil infiltration into the lung was monitored by measuring MPO activity using a previously described method (Bradley et al., J. Invest. Dermatol. 78: 206-209). Briefly, tissue samples were homogenized at 50 mg / ml in a phosphate buffer (50 mM, pH 6.0) with 0.5% hexadecyltrimethylammonium bromide. The samples were frozen, thawed three times, and centrifuged at 30,000 g for 20 minutes. The supernatants were diluted 1: 30 with an assay buffer consisting of a 50 mM phosphate buffer with pH 6.0, 0.167 mg / ml o-dianisidine (Sigma-Aldrich), and 0.0005% H202, and were spectrometrically determined the activity of the MPO enzyme in the homogenates by measuring the absorbance at 450 nm.

Bacterial load

To determine the bacterial load in the peritoneum, the peritoneal cavity was washed with 5 ml of sterile saline. A series of log solutions were made. To determine the bacterial load in the blood, 100 μΙ of blood was collected and diluted in saline. To determine the pulmonary bacterial load, the lungs were removed and equal amounts of humerus tissue homogenized and briefly centrifuged to remove thick particles. Log solutions of homogenized tissue were applied. Five hundred microliters of each solution were placed on agar blood plates and incubated for 24 hours at 37 ° C under aerobic conditions. The units that formed colonies were counted. The results were expressed as units that form a colony per milliliter or milligram of wet tissue. Determination of cytokine and pulmonary chemokine

For the determination of cytokine in tissues, protein extracts were isolated by homogenization of the lung (50 mg of tissue / ml) in 50 mM Tris-HCI, pH 7.4, with 0.5 mM DDT, and a cocktail of protease inhibitors (Roche Diagnostics). The samples were centrifuged at 3000 g for 20 minutes and stored at -80 ° C until the cytokine was determined. The levels of cytokine and chemokine were determined by a multiplex immunoassay of suspended microsphere array (Procarta Cytokine assay. Affymetrix) according to the manufacturer's recommendations and normalized to the concentration of the protein in the sample.

Extraction and quantification of protein

Frozen lung tissues were homogenized at 4 ° C in a lysis buffer containing 50 mM Tris-HCI, 5mM EDTA, 2% sodium dodecyl sulfate (SDS) and a protease inhibitor cocktail (Roche Diagnostics). The homogenized tissues were centrifuged at 5000 g at 4 ° C for 15 minutes to remove cell particles. Total protein content in the homogenized samples was extracted using Tripure® Isolation Reagent (Roche Diagnostics) according to the manufacturer's instructions. This procedure allows protein fractions to be isolated from a single sample. Protein fragments obtained using Tripure Isolation Reagent were re-suspended in 4% SDS and 8 M urea in 40 mM Tris HCI, pH 7.4, and rotated overnight at room temperature for total dissolution. The protein content of the soluble fraction was determined by spectrophotometry at 280 nm. The PCT content was analyzed by the Western blotting technique as described below, using β-actin as an internal control.

Expression of the Calc-1 gene in lung samples The expression of the CALC-1 gene was carried out as previously described (Tavares & Miñano, 2010 Clinical Science 119, (519-534) at 20 h after CLP (n = 10) rats per time point). PCT protein analysis

Western immunoblots were used using a mouse monoclonal anti-human PCT antibody (NB120-14817; Novus Biologicals) to analyze endogenous PCT in the lungs of rats. Lung samples (100 μg of total protein) were analyzed by 18% SDS-PAGE and transferred to a nitrocellulose membrane (Protran, Whatman GmbH, Dassel, Germany). The membranes were blocked for 1 hour at room temperature in a PBS-Tween buffer (0.01 M PBS and 0.1% Tween 20) containing 5% skim milk and incubated for 4 h at room temperature with the primary antibody diluted in the PBS-Tween buffer with 0.5% skim milk. The anti-PCT antibody (1: 2000; Santa Cruz Biotechnology) was used. After washing, the membrane was incubated for 1 hour at room temperature with goat anti-mouse IgG conjugated to horseradish peroxidase (Chemicon).

After rinsing, the following detection was carried out using a chemiluminescence detection kit (Thermo Scientific, Rockford, USA) according to the manufacturer's instructions. Band intensities were quantified and normalized using PCBAS 2.08e software (Raytest, Inc., Dusseldorf, Germany).

Western blotting analysis of the NF-KB nuclear translocation

Nuclear and cytoplasmic extracts were prepared for analysis by the Western blot technique of N F-κΒ p65 (nuclear) and N-F-κΒ expression inhibitor (IkBa, cytoplasmic) (mitogen activated protein kinase p44 / total p42 [MAPK] and b-actin as controls for nuclear and cytoplasmic proteins, respectively). Equal amounts of homogenized lung (50 μg of total protein) were analyzed by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Protran®, Whatman®, Whatman Gm bH, Germany). The mem branes were blocked with 5% dry skim milk (BioRAD) TPBS (0.1% Tween-20 in Phosphate Saline Buffer). After blocking, the membranes were incubated for 2 hours at room temperature with rabbit polyclonal antibodies N F-KB p65 and IkBk (1, 500; Santa Cruz Biotechnology; Santa Cruz, CA). Then, the membranes were incubated with the appropriate secondary antibody, goat anti-goat IgG (H + L) rabbit immunopuro conjugated with horseradish peroxidase, at a dilution of 1/20000 and subsequently the chemiluminescence detection method plus the Supersignal West Pico Chemiluminiscent Substrate (Thermo Scientific) detection kit was used. As internal controls, MAPK p44 / p42 anti-total antibody (for nuclear protein, diluted 1: 500; Santa Cruz Biotechnology) or anti-‐actin antibody (for cytoplasmic protein, diluted 1: 1000; Santa Cruz Biotechnology) was used. Band intensities were quantified and normalized using PCBAS, version 2.08e (Raytest Inc., Dusseldorf, Germany). Survival study

In additional groups of animals, anti-N-PCT or a vehicle was administered as described above. Twenty hours after CLP, the necrotic cecum was removed and the abdominal cavity was cleaned twice with 40 ml of normal saline, temperate and sterilized. The layered abdominal incision was then closed. The procedure for the removal of the cecum in animals that underwent CLP was carried out to simulate the clinical situation in which the septic focus is removed when possible. The animals were then allowed to eat and drink ad libitum and were monitored for 10 days to record changes in body weight and survival.

RESULTS

Alterations in the expression of the pulmonary Calc-I gene and procalcitonin As shown in Figure 1A, the pulmonary levels of CALC-I mRNA in rats subject to sepsis showed a significant increase 20 hours after the operation compared to animals. with simulated operation. When septic animals were treated with anti-N-PCT, the content of the CALC-I gene in the lungs was significantly reduced.

Effects of administration of anti-N-PCT on acute lung injury

As shown in Figure 2, rats subject to sepsis showed a significant increase in water content in the lungs compared to animals with simulated operation (P <0.01). When the septic animals were treated with anti-N-PCT, the water content in the lungs was reduced and a statistically significant difference in the water content in the lungs was not found between animals with simulated operation and those who suffered CLP and were treated with anti- N-PCT. In terms of histopathological changes, lung injury (characterized by disruption of the pulmonary architecture, extravasation of red cells, and inflammatory cells in the alveolar space) was present in animals that suffered CLP treated with a vehicle (Figure 3). Rats treated with anti-N-PCT had a large reduction of infiltrated inflammatory cells and a significant improvement in lung architecture (Figure 3). As Figure 3 indicates, the lung injury score increased significantly 20 hours after treatment with CLP and a vehicle compared to animals with simulated operation (P <0.01). Administration of anti-N-PCT after CLP significantly reduced the lung injury score by 45.4%. MPO tissue activity

The activity of the MPO lung tissue is presented in Figure 4. In the group that suffered CLP, the MPO activity of the lung tissue proved to have increased significantly (0.97 ± 0.14 versus 7.52 ± 0.49), while anti-N-PCT caused a decrease in MPO activity (3.39 ± 0.41; P <0.05). The decrease in MPO activity was in parallel with the decrease in polymophonuclear neutrophil accumulation. Although the MPO activity levels were even higher in the group treated with anti-N-PCT compared to the group that underwent simulated operation. Effects of the administration of anti-N-PCT on bacterial load

As indicated in Figures 5, the colony forming units were determined for peritoneal fluid (Figure 5A), blood (Figure 5B), and lungs (Figure 5C) in all rats 20 hours after CLP. However, the number of units that formed a colony was significantly lower in septic rats that received anti-N-PCT treatment than in those who received a vehicle treatment. Effects of the administration of anti-N-PCT on the pulmonary levels of proinflammatory cytokines

As indicated in Figure 6, the levels of TNF-α, IL-6, I L-1β and MIP-2 in the lungs increased by 29%, 1 17.4%, 214.6%, 75.78 % and 331, 3% respectively 20 hours after CLP (P <0.05). Treatment with anti-N-PCT reduced pulmonary TNF-α levels by 29.9% in animals that suffered CLP (P <0.05), and there was no significant difference in pulmonary TNF-α levels between animals with simulated operation and animals that underwent CLP and were treated with anti-N-PCT (Figure 6A). Similarly, the lung levels of IL-6, IL-β, IL-10 and MIP-2 increased 2.17 times, 3.14 times, 1.75 times and 4.3 times respectively 20 hours after CLP. The treatment with anti-N-PCT was reduced by 26.5% in IL-6 (Figure 6B), 33.58% in IL-1b (Figure 6), 68.5% in MIP-2 (Figure 6E), and IL-10 increased by 25.06% (Figure 6C).

Anti-N-PCT inhibits sepsis-induced translocation of NF-κΒ in the lungs

To study whether anti-N-PCT has any effect on the translocation of NF-κΒ in the lungs, the expression of NF-κΒ p65 in the nucleus and the expression of IkBa in the cytoplasm were determined 20 hours after CLP by a Western blot analysis. As shown in Figures 7A and 7B, the levels of NF-κΒ p65 in the nucleus increased 2.2 times 20 hours after CLP (P, 0.05). Treatment with anti-N-PCT, however, resulted in a 43.5% decrease in nuclear levels of NF-κΒ (Figure 7A) and a 30.4% increase in cytoplasmic levels of IkBa (Figure 7B). This result indicates that treatment with anti-N-PCT inhibits the degradation of IkBa and prevents translocation of NF-κΒ in the nucleus.

Effects of immunoneutralization of N-PCT on the survival rate

The survival rate after CLP and cecal excision with vehicle administration was 40% on Day 2 and decreased to 0% on Days 3 (Figure 8). The anti-N-PCT treatment, however, improved the survival rate to 80% on Day 2 and 65% on Day 3, which is significantly higher than for the group treated with the vehicle (Figure 8).

Claims

The use of an isolated peptide, or of an antibody or a fragment thereof capable of binding to N-PCT / PCT, in the preparation of a medicament for the treatment of lung lesions.

The use of an isolated peptide, or of an antibody or a fragment thereof according to claim 1, which also has the ability to inhibit the biological activity of N-PCT / PCT in vitro and / or in vivo.

The use of an isolated peptide, or of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any of claims 1-2, wherein the N-PCT / PCT is a SEQ ID sequence peptide NO: 1, the sequence peptide SEQ ID NO: 2, or a biologically active variant or fragment thereof.

The use of an isolated peptide with N-PCT / PCT binding capacity according to any of claims 1-3, wherein the peptide has been obtained by a biopanning process.

The use of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any of claims 1-3, wherein the antibody has been obtained by a method comprising: a) immunizing a mammalian animal with a peptide as described in any of claims 1-3,

 b) remove the antiserum from the animal, and optionally

 c) purify the antibody that specifically recognizes N-procalcitonin.

The use of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any of claims 1-3, wherein the antibody has been obtained by a method comprising steps (a) - (c) according to the preceding claim and, in addition, adding a cysteine at one end of a peptide of the invention before step (a).

7. The use of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any of claims 1-3, wherein the antibody has been obtained by a method comprising the steps according to any of the claims 5-6, and also comprises conjugating the peptide with KLH (Keyhole Limpet Hemocyanin) before immunization of the animal.

8. The use of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any one of claims 1-3, wherein the antibody has been obtained by a method comprising:

 a) immunizing a mammalian animal with a peptide of sequence SEQ I D NO: 1, of sequence SEQ ID NO: 2, or a biologically active variant or fragment thereof.

 b) analyze the titration against the peptide by an immunological technique, in the mammalian animal of step (a),

 c) fusing the splenocytes of host animals for the generation of immortalized cell lines,

 d) expand the clones, and optionally

 e) select the best producers.

9. The use of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any of claims 1-3, wherein the antibody has been obtained by a method comprising steps (a) - ( d) according to claim 8, and further comprises adding a cysteine at one end of a peptide of sequence SEQ ID NO: 1, of sequence SEQ ID NO:

2, or a biologically active variant or fragment thereof, before step (a).

10. The use of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any of claims 1-3, wherein the antibody has been obtained by a method comprising the steps according to any of the claims 8-9, and also comprises conjugating the peptide with KLH (Keyhole Limpet Hemocyanin) before immunization of the animal.

The use of an antibody or a fragment thereof capable of binding to the N-PCT / PCT according to any of claims 1-3, wherein the antibody has been obtained by a method comprising the steps according to any of the claims 8-10, wherein the immunological technique of step (b) is an ELISA.

12. The use of an antibody capable of binding to the N-PCT / PCT according to any of claims 1-3, obtained by a method according to any of claims 5-11, wherein the peptide of step (a) is the sequence peptide SEQ ID NO: 3, or a biologically active variant or fragment thereof.

13. The use of an antibody capable of binding to the N-PCT / PCT according to any of claims 1-3, obtained by a method according to any of claims 5-1 1, wherein the peptide of step (a) is a recombinant peptide.

14. A fusion protein comprising:

 a) a peptide according to any of claims 1-4 [peptide (i)]; and b) a transporter peptide capable of internalizing a peptide in a cell [peptide (ii)].

15. A fusion protein according to claim 14, wherein said carrier peptide comprises an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

16. The fusion protein according to claim 15, further comprising a spacer peptide located between the peptide according to any one of claims 1-4 [peptide (i)] and said carrier peptide [peptide (ii)].

17. The fusion protein according to any of claims 14-16, further comprising an amino acid sequence useful for the isolation or purification of the fusion protein of the invention.

18. A pharmaceutical composition comprising a therapeutically effective amount of a peptide and / or an antibody according to any one of claims 1-13, or a fusion protein according to any of claims 14-17, together with at least a pharmaceutically acceptable excipient.

19. The pharmaceutical composition according to claim 13, which comprises, in addition to at least one peptide and / or an antibody according to any one of claims 1-13, or a fusion protein according to any of claims 14-17, another principle active.

20. The pharmaceutical composition according to claim 19, wherein the active ingredient is a compound that inhibits or regulates the activity of NF-κΒ with anti-inflammatory activity.

21. A pharmaceutical form comprising a peptide and / or an antibody according to any of claims 1-13, or a fusion protein according to any of claims 14-17, or a pharmaceutical composition according to any of claims 18-20.

22. The use of a peptide and / or an antibody according to any of claims 1-13, of a fusion protein according to any of claims 14-17, of a pharmaceutical composition according to any of claims 18-

20, or in a pharmaceutical form according to claim 21, in the manufacture of a medicament.

23. The use of a peptide and / or an antibody according to any of claims 1-13, of a fusion protein according to any of claims 14-17, of a pharmaceutical composition according to any of claims 18-20, or in a pharmaceutical form according to claim

21, in the development of a medication for the treatment of lung lesions.

24. The use of a peptide and / or an antibody according to any of claims 1-13, of a fusion protein according to any of claims 14-17, of a pharmaceutical composition according to any of claims 18-20, or in a pharmaceutical form according to claim 21, in the preparation of a medicament for the treatment of indirect lung lesions. 25. The use of a peptide and / or an antibody according to any of the claims

1-13, of a fusion protein according to any of claims 14-17, of a pharmaceutical composition according to any of claims 18-20, or of a pharmaceutical form according to claim 21, in the preparation of a medicament for the treatment of acute lung lesions.

26. The use of a peptide and / or an antibody according to any of claims 1-13, of a fusion protein according to any of claims 14-17, of a pharmaceutical composition according to any of claims 18-20, or of a pharmaceutical form according to claim 21, in the preparation of a medicament for the treatment of acute respiratory distress syndrome.

27. The use of a peptide according to any of claims 1-13, of a fusion protein according to any of claims 14-17, of a pharmaceutical composition according to any of claims 18-20, or of a pharmaceutical form according to the claim 21, in the preparation of a medicament for the treatment of sepsis-derived lung injury.

28. The use of a peptide according to any of claims 1-13, of a fusion protein according to any of claims 14-17, of a pharmaceutical composition according to any of claims 18-20, or of a pharmaceutical form according to the claim 21, in the preparation of a medicament for the treatment of septic shock.