Immunomodulatory peptide

Abstract

Embodiments of the present disclosure relate to an isolated peptide consisting of a sequence of 3 to 39 amino acids derived from the amino acid sequence SEQ ID NO: 1, said peptide having a sequence of amino acids selected from the group consisting of: a) sequences of 3 to 39 amino acids comprising at least the residues 6 to 8 of SEQ ID NO: 1, and b) sequences of 3 to 39 amino acids having at least 70% identity with said sequence in a).

Claims

1. A method of treating a subject with a condition responsive to activated cytotoxic functions of macrophages and characterized by expression of molecular patterns recognized by C-type lectin receptors, the method comprising administering to the subject a therapeutically effective amount of a medicament comprising a peptide, the peptide only consisting of a sequence of amino acids selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, wherein the peptide is amidated at the C-terminal and wherein the peptide is a pro-inflammatory agent that increases expression of C-type lectin receptors on the surface of macrophages, thereby activating the cytotoxic functions of the macrophages when in contact with molecular patterns characterizing the condition.

2. The method according to claim 1, wherein said peptide consists of the amino acid sequence SEQ ID NO: 1.

3. The method of claim 1, wherein the condition is caused by a pathogen recognised by a C-type lectin receptor.

4. The method of claim 1, wherein the condition is mycosis.

5. The method of claim 1, wherein the condition is cancer with tumour cells having glycan patterns recognized by C-type lectin receptors.

6. The method of claim 3, wherein the condition is caused by a pathogen recognised by a mannose or dectin-1 receptor.

7. The method of claim 4, wherein the condition is candidiasis.

8. The method according to claim 1, wherein the peptide has a sequence of amino acids selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

9. A method of activating a cytotoxic phenotype in macrophages, comprising contacting the macrophages with an effective amount of a medicament comprising a peptide, the peptide only consisting of a sequence of amino acids selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, wherein the peptide is amidated at the C-terminal, and wherein the peptide is a pro-inflammatory agent that increases expression of C-type lectin receptors on the surface of the macrophages, whereby the cytotoxic functions of the macrophages are activated when in contact with molecular patterns recognized by C-type lectin receptors.

10. The method according to claim 9, wherein said peptide consists of the amino acid sequence SEQ ID NO: 1.

11. The method according to claim 9, wherein the method is a method for treating a disease in a subject caused by a pathogen recognised by a C-type lectin receptor, wherein contacting the macrophages with an effective amount of the medicament comprises administering to the subject a therapeutically effective amount of the medicament.

12. The method of claim 11, wherein the pathogen is recognised by a mannose or dectin-1 receptor.

13. The method according to claim 11, wherein the disease is mycosis.

14. The method of claim 11, wherein the disease is candidiasis.

15. The method according to claim 9, wherein the method is a method for treating of cancer in a subject characterized by tumour cells having glycan patterns recognized by C-type lectin receptors, wherein contacting the macrophages with an effective amount of the medicament comprises administering to the subject a therapeutically effective amount of the medicament.

16. The method according to claim 9, wherein the peptide has a sequence of amino acids selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

Description

FIGURES

(1) FIG. 1 shows the removal of C. albicans by macrophages derived from human monocytes treated, or untreated, by the in vitro P17.

(2) FIG. 2 shows the binding and phagocytic capacity of C. albicans by macrophages derived from human monocytes treated or untreated by the P17.

(3) FIG. 3 shows the effect of P17 on the production of oxygen free radicals (OFR) and cytokines, such as IL-1β, TNFα, IL-10 and IL-12, by macrophages derived from human monocytes in response to a stimulation by C. albicans.

(4) FIG. 4 shows the effect of P17 on the protein expression of C-type lectin receptors.

(5) FIG. 5 shows the effect of P17 on the expression of marker genes of the conventional and alternative differentiation of the macrophages.

(6) FIG. 6 shows the effect of P17 on the expression of encoding genes PPAR-γ and the target SRB1 thereof, and on the expression of membrane receptors Dectin-1 and MR in the presence of GW9662, an irreversible antagonist of PPAR-γ in macrophages derived from human monocytes.

(7) FIG. 7 shows the effect of P17 on the expression of genes encoding enzymes of the metabolism of arachidonic acid in macrophages derived from human monocytes.

(8) FIG. 8 shows the effect of P17 on the secretion of LTB4 by macrophages derived from human monocytes.

(9) FIGS. 9A-C represent the intracellular calcium concentration induced by P17 with respect to negative tests (A), in the presence of pertussis toxin (B) and by desensitising with fMLP (peptide N-Formylmethionine-leucyl-phenylalanine) (C).

(10) FIG. 10 shows the effect of BAPTA (calcium binder) on the antimicrobial effect of P17, the release of reactive derivatives of oxygen (ROS) and of IL-1β.

(11) FIG. 11 shows the effects of administering in vitro P17 on the body weight of mice having a gastrointestinal candidiasis induced by C. albicans.

(12) FIG. 12 shows the effect of administering in vivo P17 on the gastrointestinal colonisation, 6 days post-infection.

(13) FIGS. 13A-E show the effect of administering P17 in mice infected over the capacity of the peritoneal macrophages thereof (A-B) to remove and phagocytose Candida albicans, (C) to produce ROS, and (D-E) to release IL-β and IL-12.

(14) FIG. 14 illustrates the mode of action of P17.

(15) FIG. 15 shows the number of T lymphoma cells (EL4) in co-culture with macrophages treated or untreated by P17.

(16) FIG. 16 shows the ROS production capacity of macrophages treated, or untreated, by P17 in response to EL4 tumour cells.

(17) FIG. 17 shows the effect of different fragments of P17 on the release of ROS by macrophages derived from human monocytes in response to C. albicans.

(18) FIG. 18 shows the effect of different fragments of P17 on the removal of Candida albicans by macrophages derived from human monocytes.

(19) FIG. 19 shows the effect of C-terminal amidation of P17 on the production of ROS by macrophages in response to C. albicans.

EXAMPLES

(20) Equipment and Methods:

(21) P17 Anti-Microbial Peptide

(22) Unless it is specified otherwise, the peptide P17 used in the figures and the examples below is amidated at the C-terminal (presence of an NH.sub.2 group at the C-terminal end thereof). The sequence thereof has been characterised by de novo sequencing by using mass spectrometry and Edman degradation (Rifflet et al. 2012). The peptide P17 has been synthesised on an automated Liberty peptide synthesiser (CEM, Saclay, France) with a purity greater than 99%. The molecular authenticity and identity of synthetic peptides have been tested by MALDI-TOF-MS.

(23) Study of Removing C. albicans by Macrophages Derived from Human Monocytes

(24) The macrophages derived from human monocytes have been treated, or not, with the peptide P17 or fragments of it then incubated at 37° C. for 24 hours. These cells have been incubated for 40 minutes at 37° C. with C. albicans (at a ratio of 0.3 yeasts per macrophage) and the non-bound yeasts are removed by washing. These macrophages are thus incubated at 37° C. for 4 hours. After incubation, the environment has been removed and the cells have been lysed. The units forming colonies (CFU) of C. albicans have been determined on Sabouraud boxes after spreading and culturing for 48 hours at 37° C.

(25) Study of Binding and of the Phagocytosis of C. albicans by Macrophages Derived from Human Monocytes

(26) To analyse the binding and the phagocytosis of C. albicans, macrophages derived from human monocytes have been treated, or not, with the peptide P17, then incubated at 37° C. for 24 hours. The macrophages derived from human monocytes have been brought together with the yeasts marked GFP (ratio 1/6) then incubated at 4° C. for 20 minutes, in order to evaluate the binding. The phagocytosis has been evaluated after 1 hour of incubation at 37° C. The quantity of C. albicans bound or ingested by macrophages derived from human monocytes has been determined by measuring the fluorescence by using a fluorimeter (PerkinElmer EnVision).

(27) Study of the Production of ROS by Macrophages Derived from Human Monocytes in Response to C. albicans

(28) To analyse the production of ROS, the macrophages derived from human monocytes have been treated, or not, with the peptide P17, P17, non-amidated, or with the following P17 fragments, P17(1-5), P17(1-7), P17(1-9), P17(1-11), P17(3-13), P17(5-13), P17(7-13) and P17(9-13); the fragments P17(1-5), P17(1-7), P17(1-9), P17(1-11) not being amidated at the C-terminal and the fragments P17(3-13), P17(5-13), P17(7-13) and P17(9-13) being amidated at the C-terminal. Then, the macrophages derived from human monocytes thus treated have been incubated at 37° C. for 24 hours. The production of reactive derivatives of oxygen (ROS) has been measured by chemiluminescence in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) (EnVision; PerkinElmer). The chemiluminescence generation has been continually measured for 1 hour 30 minutes after incubation of cells with luminol (66 μM) in the presence, or not, of C. albicans (yeast ratio per macrophage: 3:1).

(29) Study of Production of ROS by Murine Macrophages in Response to Murine Lymphoma T Tumour Cells (EL4)

(30) To analyse the production of ROS, peritoneal murine macrophages have been treated, or not, with the peptide P17 then incubated at 37° C. for 24 hours. The production of reactive derivatives of oxygen (ROS) has been measured by chemiluminescence in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) (EnVision; PerkinElmer). The chemiluminescence generation has been continually measured for 1 hour 30 minutes after incubation of cells with luminol (66 μM) in the presence, or not, of EL4 lymphoma T tumour cells.

(31) Study of the Production of IL-1β, TNFα, IL-10 and IL-12 by Macrophages Derived from Human Monocytes in Response to C. albicans

(32) Macrophages derived from human derivatives have been stimulated with the peptide P17 for 24 hours, then these macrophages activated by the peptide P17 have been stimulated with C. albicans at a yeast per macrophage ratio of 3:1 for 8 hours. The production of IL-Iß, TNFα, IL-10 and IL-12 in cell supernatants has been determined by ELISA (OptiEIA BD Biosciences).

(33) Evaluation of the Protein Expression of C-Type Lectin Receptors

(34) To analyse the protein level of C-type lectin receptors, the macrophages derived from human monocytes have been treated, or not, with the peptide P17 (200 μg/ml) then incubated at 37° C. for 24 hours. The cells collected have been centrifugated at 1500 rpm for 10 minutes and nerve cells have been suspended in the PBS supplemented with 1% foetal calf serum (FCS). The cells have been marked with antibodies coupled with different fluorochromes directed against the Dectin-1 receptor (mAb; R&D FAB1859C-100, 1/40), DC-SIGN (mAb; BD Biosciences 551 265, 1/20) CD16 (mAb; PNIM 0814, 1/20), CD36 (mAb; BD Biosciences 550 956, 1/40) and the mannose receptor (specific MR (mannose receptor) ligand) conjugated FITC (Sigma A7790, 1 mg/ml 1/100)). The analysis has been carried out by flow cytometry (Becton Dickinson FACScalibur) over a population of 10000 cells.

(35) Reverse Transcription and PCR in Real Time

(36) Macrophages derived from human monocytes have been treated with P17 (200 μg/ml) for 8 hours. The preparation of DNA has been carried out by using a kit (EZ-10 Spin Column Total RNA Minipreps Super Kit by Bio Basic) and by following the instructions of the manufacturer. The synthesis of cDNA has been carried out according to the instructions of the manufacturer (Thermo Electron). An RT-qPCR has been produced on a LightCycler 480 system by using the LightCycler SYBR Green I Master (Roche Diagnostics). Primers (Eurogentec) have been produced with the software Primer 3. The mRNA of GAPDH has been used as a control. Pooled cDNA samples diluted in series have been used as external standard in each series for the quantification.

(37) The sequences of primers are given in the table below.

(38) TABLE-US-00002 TABLE Sequences of human primers used in qPCR analysis Gene Sequence 5′-3′ Alox5 antisense ACT-GGA-AAC-ACG-GCA-AAA-AC (SEQ ID NO: 18) sense     TTT-CTC-AAA-GTC-GGC-GAA-GT (SEQ ID NO: 19) Itgam antisense TTG-CAT-CCA-TCT-CAA-ATC-CA (SEQ ID NO: 20) (CD11b) sense     CTC-CCA-AAG-TGC-TGG-GAT-TA (SEQ ID NO: 21) Fcgr3 antisense TAC-AGC-GTG-CTT-GAG-AAG-GA (SEQ ID NO: 22) (CD16) sense     GCA-CCT-GTA-CTC-TCC-ACT-GT (SEQ ID NO: 23) Fcgr2 antisense CCA-AAG-GCT-GTG-CTG-AAA-CT (SEQ ID NO: 24) (CD32) sense     TAC-TCC-CCG-CTG-TCA-TTG-TT (SEQ ID NO: 25) Cd36 antisense TGA-TAG-GTG-CAG-CAA-AGC-AC (SEQ ID NO: 26) sense     TGT-AAC-CCA-GGA-CGC-TGA-GG (SEQ ID NO: 27) Clec7a antisense CCA-AGC-ATA-GGA-TTC-CCA-AAA (SEQ ID NO: 28) (Dectin-1) sense     AAA-AGG-ATC-GTG-TGC-TGC-ATC (SEQ ID NO: 29) Ptgs2 antisense TGA-GCA-TCT-ACG-GTT-TGC-TG (SEQ ID NO: 30) (COX-2) sense     TGC-TTG-TCT-GGA-ACA-ACT-GC (SEQ ID NO: 31) Pla2g4a antisense GCC-TTG-GTG-AGT-GAT-TCA-GCT (SEQ ID NO: 32) (cPLA2) sense     AGA-TTC-AAG-CCC-AGC-ATG-AAG (SEQ ID NO: 33) Cd209 antisense GGG-CAT-GGA-GGC-TCC-AC (SEQ ID NO: 34) (DC-SIGN) sense     CAA-CTT-AGA-AAC-AGC-CAA-ATG-GAA (SEQ ID NO: 35) Alox5ap antisense ACC-CGC-TCA-AAG-GCA-ATG-G (SEQ ID NO: 36) (FLAP) sense     CAC-GAA-AGC-AGG-ACC-CAG-A (SEQ ID NO: 37) Gapdh antisense AGG-TCG-GAG-TCA-ACG-GAT-TT (SEQ ID NO: 38) sense     ATC-TCG-CTC-CTG-GAA-GAT-GG (SEQ ID NO: 39) Il1b antisense CAG-CCA-ATC-TTC-ATT-GCT-CA (SEQ ID NO: 40) sense     AGG-CAG-AGA-GGG-AAG-GAG-AG (SEQ ID NO: 41) Il6 antisense TAC-CCC-CAG-GAG-AAG-ATT-GT (SEQ ID NO: 42) sense     TTT-TCT-GCC-AGT-GCC-TCT-TT (SEQ ID NO: 43) Il12 antisense TGG-GTG-GGT-CAG-GTT-TGA-TG (SEQ ID NO: 44) sense     GCC-CAG-CTG-CTG-AGG-AGA-GT (SEQ ID NO: 45) Il10 antisense TGC-AAA-ACC-AAA-CCA-CAA-GA (SEQ ID NO: 46) sense     TCT-CGG-AGA-TCT-CGA-AGC-AT (SEQ ID NO: 47) Il1ra antisense TGG-GAA-TCT-CAG-ATG-GGA-AG (SEQ ID NO: 48) sense     CTG-TGT-CCC-CCA-GAA-CTT-GT (SEQ ID NO: 49) Tgfb1 antisense ACT-GAG-GGG-AAG-GGA-CAA-CT (SEQ ID NO: 50) sense     TCG-GTA-CCA-GGT-GAG-GGT-AG (SEQ ID NO: 51) p47.sup.phox antisense CCT-CAT-TGT-CCA-GTG-TGG-TG (SEQ ID NO: 52) sense     TCT-TCC-GTC-TCG-TCA-GGA-CT (SEQ ID NO: 53) Lta4h antisense ACT-GCT-TGG-AGG-ACC-AGA-GA (SEQ ID NO: 54) sense     GGA-AAG-CAT-TAG-CAG-GCA-AG (SEQ ID NO: 55) Mrc-1 (MR) antisense GGC-GGT-GAC-CTC-ACA-AGT-AT (SEQ ID NO: 56) sense     ACG-AAG-CCA-TTT-GGT-AAA-CG (SEQ ID NO: 57) Ptges antisense CAT-GTG-AGT-CCC-TGT-GAT-GG (SEQ ID NO: 58) (PGES) sense     GAC-TGC-AGC-AAA-GAC-ATC-CA (SEQ ID NO: 59) Pparg antisense GCT-GTG-CAG-GAG-ATC-ACA-GA (SEQ ID NO: 60) sense     GGG-CTC-CAT-AAA-GTC-ACC-AA(SEQ ID NO: 61) Tnfα antisense TCC-TTC-AGA-CAC-CCT-CAA-CC (SEQ ID NO: 62) sense     AGG-CCC-CAG-TTT-GAA-TTC-TT (SEQ ID NO: 63)
Determination of the Intracellular Calcium Concentration

(39) The intracellular calcium concentration has been measured by using a Fluo 3-AM fluorescent probe (Molecular Probe). Macrophages derived from human monocytes (1.5×10.sup.5) have been incubated with 11.5×10.sup.−6 M of Fluo 3-AM for 30 minutes at 37° C. The intracytosolic Ca2+ level has been recorded every 0.5 seconds for a total period of 3 minutes after the addition of P17 (200 μg/ml). In desensitisation experiments, a second injection of N-Formylmethionine-leucyl-phenylalanine (fMLP) bacterial peptide of P17 has been carried out at the end of the recording of the fluorescence and the level of intracytosolic Ca2+ level recorded for 3 minutes in addition. In certain experiments, macrophages derived from human monocytes have been preincubated with U73122 (2 μM) or HBSS without calcium for 10 minutes before the addition of P17. The fluorescence has been quantified by using the approach based on EnVision fluorimetry (PerkinElmer).

(40) Experimental Gastrointestinal Candida Murine Model

(41) For in vivo experiments, a gastrointestinal infection with the C. albicans strain 98/26135 has been established by feeding with 50×10.sup.6 of C. albicans per mouse (n=8 per group). No antibiotic or immunosuppressant treatment has been used to facilitate the infection of mucous membranes of the oral cavity and of the gastrointestinal tract. The mice have been treated intraperitoneally (i.p.) with the peptide P17 (10 μg per mouse) or with a saline solution for control groups, 1 day before the injection with C. albicans, 1 day after infection and then every 2 days (5 injections). The body weight of each mouse has been recorded daily. On the 8.sup.th day after treatment (7 days after infection), all mice have been euthanised by asphyxiation with CO.sub.2.

(42) The oesophagus, the stomach, the caecum and the liver have been removed aseptically to evaluate the colonisation by C. albicans. The peritoneal macrophages of the mice have been sampled and the ex vivo capacities thereof to remove and to phagocytose Candida albicans have been determined. The production of ROS, IL-β and IL-12 by these macrophages in response to Candida albicans have also been evaluated.

(43) Quantification of the Number of Lymphoma T Tumour Cells (EL4) in the Presence of Murine Macrophages

(44) Peritoneal murine macrophages have been treated, or not, with the peptide P17 then incubated at 37° C. for 24 hours. These macrophages have been incubated for 40 minutes at 37° C. with luminescent lymphoma T cells (EL4-luc) and unbound cells are removed by washing. These macrophages are thus incubated at 37° C. for 4 hours. After incubation, the number of lymphoma T cells (EL4-luc) is evaluated by chemiluminescence.

(45) Results:

(46) FIG. 1 shows that the presence of P17 considerably increases the capacity of human macrophages to remove in vitro C. albicans.

(47) These results have been confirmed in vivo on mice infected by C. albicans. Indeed, mice having a gastrointestinal candidiasis treated by P17 would have a lesser loss of weight with respect to untreated mice (FIG. 11). Consistently, the gastrointestinal colonisation by Candida albicans was significantly lower in mice treated by P17 with respect to untreated mice (FIG. 12).

(48) The inventors have sought to understand the mechanism underlying the action of P17.

(49) The macrophages derived from human monocytes treated by P17 both have a greater capacity to recognise and to phagocytose C. albicans (FIG. 2). In addition, P17 induces a higher production of ROS in response to stimulation by C. albicans and pro-inflammatory cytokines such as IL-113 and TNFα (FIG. 3). The increase of producing these cytotoxic mediators by the peptide P17 is associated with the overexpression of C-type lectin receptors on the surface of macrophages derived from human monocytes (FIG. 4).

(50) P17 Improves the Anti-Tumour Response of the Macrophages

(51) As FIG. 15 shows, in the presence of macrophages polarised by P17, the number of EL4 tumour cells decreases significantly. These results reveal that the macrophages polarised by P17 have a significant cytotoxic activity regarding tumour cells, with respect to untreated microphages.

(52) This induction of cytotoxic activity of the macrophages by P17 regarding tumour cells is correlated with an increased production of ROS (FIG. 16).

(53) Effects of P17 Fragments on the Production of Reactive Species of Oxygen (ROS) and on the Removal of Candida albicans by Macrophages

(54) The capacity to induce the release of ROS in response to C. albicans of macrophages treated by different P17 fragments has been tested.

(55) The results obtained with the peptides which lack the C-terminal end (P17(1-5), P17(1-7), P17(1-9), P17(1-11)) shows the importance of this region in the activation of the cytotoxic functions of the macrophages by P17 (FIGS. 17-18). Indeed, the treatment of macrophages with these fragments does not induce the production of ROS, nor the removal of Candida albicans.

(56) The peptides P17(1-5), P17(1-7), P17(1-9), P17(1-11) tested were not amidated at the C-terminal contrary to P17. The difference of ROS release between amidated P17 and non-amidated P17 has therefore been tested in order to determine the impact of the C-terminal amidation on the pro-inflammatory properties of P17. These results show that the C-terminal amidation of P17 is essential to the pro-inflammatory activity thereof (FIG. 19).

(57) The ROS release induced by the fragments from which the N-terminal portion has been deleted (P17(3-13), P17(5-13), P17(7-13), P17(9-13) has also been tested. Only the fragments P17(3-13) and P17(5-13) conserve the activity thereof (FIG. 17). These results show the major role of central amino acids in the immunomodulatory activity of P17.

(58) The effects of the different fragments on the removal of C. albicans by the macrophages have also been tested. The results are identical to those obtained for the release of ROS. Fragments with no C-terminal portion are inactive, while the fragments P17(3-13) and P17(5-13) have an activity close to that of P17.