TREM-1 INHIBITOR FOR USE IN THE TREATMENT OF A SUBJECT SUFFERING FROM A CORONAVIRUS INFECTION

Abstract

An inhibitor of triggering receptor expressed on myeloid cells 1 (TREM-1) for use in the treatment of coronavirus disease 2019 (COVID-19) in a subject in need thereof, in particular in a subject suffering from a severe form and/or a complication of COVID-19. Also, the use of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as a marker in a method for identifying a subject suffering from COVID-19 susceptible to respond to a TREM-1 inhibitor and in a method for monitoring the effectiveness of TREM-1 inhibitor administered to a subject suffering from COVID-19.

Claims

1-14. (canceled)

15. A method for treating coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof, said method comprising administering to the subject a triggering receptor expressed on myeloid cells-1 (TREM-1) inhibitor.

16. The method according to claim 15, wherein the subject is suffering from a severe form and/or at least one complication of COVID-19.

17. The method according to claim 16, wherein the at least one complication of COVID-19 is selected from the group consisting of respiratory failure, including acute respiratory failure or acute respiratory distress syndrome (ARDS); persistence of respiratory failure including the requirement for prolonged mechanical ventilation or failed extubation; secondary infection or superinfection; thrombotic complications including venous and/or arterial thromboembolism; pulmonary embolism; cardiocirculatory failure (also referred to as cardiovascular failure); renal failure; liver failure; and any combinations thereof.

18. The method according to claim 15, wherein the TREM-1 inhibitor is administered by intravenous infusion at a dose ranging from about 0.1 mg/kg/h to about 3 mg/kg/h.

19. The method according to claim 15, wherein the TREM-1 inhibitor is administered by intravenous infusion at a dose ranging from about 0.3 mg/kg/h to about 1 mg/kg/h.

20. The method according to claim 15, wherein the TREM-1 inhibitor is selected from the group consisting of peptides inhibiting the function, activity and/or expression of TREM-1; antibodies directed to TREM-1, soluble TREM-1 (sTREM-1), TREM-1 ligand and/or sTREM-1 ligand; small molecules inhibiting the function, activity and/or expression of TREM-1; siRNAs directed to TREM-1; shRNAs directed to TREM-1; antisense oligonucleotide directed to TREM-1; ribozymes directed to TREM-1; and aptamers directed to TREM-1.

21. The method according to claim 15, wherein the TREM-1 inhibitor is a peptide inhibiting the function, activity and/or expression of TREM-1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, or comprising an amino acid sequence with at least 80% identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

22. The method according to claim 15, wherein the TREM-1 inhibitor is a peptide inhibiting the function, activity and/or expression of TREM-1 comprising an amino acid sequence as set forth in SEQ ID NO: 10 or comprising an amino acid sequence with at least 80% identity with SEQ ID NO:10.

23. An in vitro method for identifying a subject suffering from coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptible to respond to a triggering receptor expressed on myeloid cells-1 (TREM-1) inhibitor, said method comprising: measuring the level of soluble TREM-1 (sTREM-1) in a biological sample from the subject; and comparing the level of sTREM-1 measured in the biological sample from the subject to a reference value.

24. The in vitro method according to claim 23, wherein the subject is suffering from a severe form and/or at least one complication of COVID-19.

25. The method according to claim 15, wherein the subject to be treated is identified according to an in vitro method for identifying a subject suffering from coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptible to respond to a triggering receptor expressed on myeloid cells-1 (TREM-1) inhibitor, said method comprising: measuring the level of soluble TREM-1 (sTREM-1) in a biological sample from the subject; and comparing the level of sTREM-1 measured in the biological sample from the subject to a reference value

26. An in vitro method for monitoring the effectiveness of a triggering receptor expressed on myeloid cells-1 (TREM-1) inhibitor administered to a subject suffering from coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), said method comprising: measuring the level of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in a biological sample from the subject; and comparing the level of sTREM-1 measured in the biological sample from the subject to a reference value.

27. The in vitro method according to claim 26, wherein the reference value is a personalized reference value of the subject.

28. The in vitro method according to claim 27, wherein the personalized reference value of the subject is the level of sTREM-1 measured in a sample obtained from the subject before or at the beginning of the administration of the TREM-1 inhibitor.

29. The in vitro method according to claim 26, wherein the subject is suffering from a severe form and/or at least one complication of COVID-19.

30. The in vitro method according to claim 26, wherein the TREM-1 inhibitor is a peptide inhibiting the function, activity and/or expression of TREM-1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, or comprising an amino acid sequence with at least 80% identity with SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0295] FIG. 1 is a Kaplan-Meier graph showing the survival over 10 days of transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h. N=16 mice per treatment group. P value calculated by a Log rank test.

[0296] FIG. 2 is a graph showing the evolution of the bodyweight over 10 days of transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h. N=16 mice per treatment group. Data are represented as mean and SEM (standard error to the mean). The last observation carried forward (LOCF) approach was used to impute missing data. P value was calculated by a mixed-effect analysis.

[0297] FIG. 3 is a graph showing the bodyweight loss over 10 days of transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h. N=16 mice per treatment group. Data are represented as mean and SEM (standard error to the mean). The last observation carried forward (LOCF) approach was used to impute missing data. P value was calculated by a mixed-effect analysis.

[0298] FIG. 4 is a graph showing the clinical score over 10 days of transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h. The clinical score was assessed on a scale of 1 (healthy mouse) to 5 (dead mouse), with a lower score corresponding to a better clinical status. N=16 mice per treatment group. Data are represented as mean and SEM (standard error to the mean). P value was calculated by a mixed-effect analysis.

[0299] FIG. 5 is a graph showing the respiratory score over 10 days of transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h. The respiratory score was assessed on a scale of 0 (normal, rapid mouse respiration) to 5 (dead mouse), with a lower score corresponding to a better respiratory function. N=16 mice per treatment group. Data are represented as mean and SEM (standard error to the mean). P value was calculated by a mixed-effect analysis.

[0300] FIG. 6 is a graph showing sTREM-1 plasma concentration over time for groups 1-4 (at 24 and 72 hours after infection for groups 1 and 2, and at 48 and 96 hours after infection for groups 3 and 4) of transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h. N=8 mice per group. Data are represented as box and whiskers plot from min to max and showing all points. p values were calculated with a non-parametric t-test comparing LR12-treated and vehicle-treated conditions.

[0301] FIGS. 7A-J are a set of graphs showing the plasma concentrations over time for groups 1-4 (at 24 and 72 hours after infection for groups 1 and 2, and at 48 and 96 hours after infection for groups 3 and 4) of the following cytokines/chemokines: interferon γ or IFNγ (FIG. 7A), keratinocyte chemoattractant or KC (FIG. 7B), monocyte chemoattractant protein 1 or MCP-1 (FIG. 7C), interleukin-12 (active heterodimer) or IL12p70 (FIG. 7D), Regulated on Activation, Normal T cell Expressed and Secreted or RANTES (FIG. 7E), interferon gamma-induced protein 10 or IP-10 (FIG. 7F), interleukin-10 or IL-10 (FIG. 7G), interferon α or IFN-α (FIG. 7H), interleukin-6 or IL-6 (FIG. 7I), and granulocyte-macrophage colony-stimulating factor or GM-CSF (FIG. 7J) of transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h. N=8 mice per group. Data are represented as box and whiskers plot from min to max and showing all points. p values were calculated with a non-parametric t-test comparing LR12-treated and vehicle-treated conditions at different timepoints.

[0302] FIGS. 8A-O are a set of graphs showing the number of CD45.sup.+ cells (FIG. 8A), eosinophils (FIG. 8B), monocytes-derived macrophages (FIG. 8C), interstitial macrophages (FIG. 8D), alveolar macrophages (FIG. 8E), neutrophils (FIG. 8F), CD103.sup.+ dendritic cells (FIG. 8G), monocytes-derived dendritic cells (FIG. 8H), conventional dendritic cells (FIG. 8I), CD11b.sup.+ dendritic cells (FIG. 8J), NK cells (FIG. 8K), CD8.sup.+ lymphocytes (FIG. 8L), CD4.sup.+ lymphocytes (FIG. 8M), NKT cells (FIG. 8N), plasmacytoid dendritic cells (FIG. 8O) in lungs harvested 48 hours after infection from transgenic mice expressing the human SARS-CoV-2 receptor (K18 ACE2) infected with SARS-CoV-2 at t.sub.0 and administered by intraperitoneal injection with 2 mg of TLT-1 peptide LR12 or vehicle (NaCl 0.9%) at t.sub.0+1 h, t.sub.0+24 h, and t.sub.0+48 h. N=8 mice per group. Data are represented as mean and SEM (standard error to the mean). P values are calculated by unpaired parametric t-test.

EXAMPLES

[0303] The present invention is further illustrated by the following examples.

Example 1: Mouse Model of SARS-CoV-2 Infection

Materials and Methods

Material

[0304] Mice: transgenic male mice B6.Cg-Tg(K18-ACE2)2Prlmn/J, 7-week-old at arrival, were obtained from Charles River (The Jackson Laboratory). The transgenic B6.Cg-Tg(K18-ACE2)2Prlmn/J mice express the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) and are susceptible to SARS-CoV-2 pulmonary infection (Yinda et al. K18-hACE2 mice develop respiratory disease resembling severe COVID-19. PLoS Pathog. 2021 Jan. 19; 17(1):e1009195). The animals were housed in ventilated and enriched plastic cages containing irradiated sawdust as a bedding material, as prescribed by the housing standards throughout the experimental phase. Mice were housed in groups of maximum 5 animals per cage on a regular light-dark cycle, 22±2° C. and at 50±10% relative humidity. During the acclimation phase and experimental phase, standard diet (RM1 (E) 801492, SDS) and tap water were provided ad libitum. All procedures performed on animals in the course of the study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). All the in vivo protocol design and procedures were approved by an Ethical Committee under ethical protocol 2020101616517580_v1 #27729.

[0305] SARS-CoV-2 strain: SARS-CoV-2 was isolated from a patient with laboratory-confirmed COVID-19 in Toulouse, France. Compared to the sequence of the Wuhan reference strain, an alignment showed 99.96% identity between the two strains. One notable variation between the two strains is a glycine (Gly) at position 614 in Spike protein of the Toulouse strain, vs. an aspartic acid (Asp) at the same position in the Wuhan reference strain.

[0306] TLT-1 peptide: mouse LR12 (LQEEDTGEYGCV—SEQ ID NO: 20), the murine equivalent of human LR12 (LQEEDAGEYGCM—SEQ ID NO: 10), was provided as a −80° C. frozen stock solution at 40 mg/mL in a phosphate-citrate-arginine buffer. Solution for administration was extemporaneously prepared by thawing out an aliquot of stock solution at room temperature and by diluting it 4-times in physiological serum (NaCl 0.9%). Each mice will then receive 2 mg of peptide at each injection.

[0307] Vehicle: phosphate-citrate-arginine buffer was provided as vehicle, as a −80° C. frozen stock solution. Solution for administration was extemporaneously prepared by thawing out an aliquot of stock solution at room temperature and by diluting it 4-times in physiological serum (NaCl 0.9%).

Methods

[0308] Infection: on day 0 at to, the mice were infected with 25 μL of DMEM (Dulbecco's Modified Eagle Medium) containing SARS-CoV-2 (SARS-CoV-2 strain isolated in Toulouse. Compared to the sequence of Wuhan reference strain, an alignment shows 99.96% identity between the two strains. One notable mutation is a GLY at position 614 in Spike protein, vs an ASP in Wuhan strain at the same position) through intranasal route (2.5×10.sup.3 PFU/mouse).

[0309] Treatment: following infection, the mice were administered by intraperitoneal (i.p.) injection either 200 μL of murine LR12 peptide (at a concentration of 10 mg/mL, i.e., 2 mg corresponding to a dose of 80 mg/kg for a mouse of 25 grams) or 200 μL of vehicle at t.sub.0+1 h, t.sub.0+24 h, t.sub.0+48 h, t.sub.0+72 h and t.sub.0+96 h.

Mice were thus assigned to each of the following groups: [0310] Group 1 (8 mice)—infection with 2.5×10.sup.3 PFU/mouse+vehicle: daily clinical signs and blood sampling at 24 and 72 hours; [0311] Group 2 (8 mice)—infection with 2.5×10.sup.3 PFU/mouse+2 mg LR12: daily clinical signs and blood sampling at 24 and 72 hours; [0312] Group 3 (8 mice)—infection with 2.5×10.sup.3 PFU/mouse+vehicle: daily clinical signs and blood sampling at 48 and 96 hours; [0313] Group 4 (8 mice)—infection with 2.5×10.sup.3 PFU/mouse+2 mg LR12: daily clinical signs blood sampling at 48 and 96 hours; [0314] Group 5 (8 mice)—infection with 2.5×10.sup.3 PFU/mouse+vehicle: lung harvest at 48 h; and [0315] Group 6 (8 mice)—infection with 2.5×10.sup.3 PFU/mouse+2 mg LR12: lung harvest at 48 h.

[0316] Sample collection: for plasma production and cytokine analyzes, on day 1 (D1) and day 3 (D3), blood was collected from the mice of groups 1 and 2; and on day 2 (D2) and day 4 (D4), blood was collected from the mice of groups 3 and 4. Blood sampling was done before the treatment. On D2, lungs were harvested from the mice of groups 5-6 for flow cytometry analyzes.

[0317] Monitoring: from day 0 (D0) to day 10 (D10), the bodyweight and survival of all mice from groups 1 to 4 were monitored. From D0 to D10, the clinical score of the mice from groups 1 to 4 was recorded on a scale from 1 to 5 defined as follows: 1=healthy mouse; 2=mouse showing signs of malaise, including slight piloerection, slightly changed gait and increased ambulation; 3=mouse showing signs of strong piloerection, constricted abdomen, changed gait, periods of inactivity; 4=mouse with enhanced characteristics of the previous groups, but showing little activity and becoming moribund; 5=dead mouse. From D0 to D10, the clinical score of the mice from groups 1 to 4 was recorded on a scale from 0 to 5 defined as follows: 0=normal, rapid mouse respiration; 1=slightly decreased respiration; 2=moderately reduced respiration; 3=severely reduced respiration; 4=asphyxia; 5=dead mouse.

[0318] Flow cytometry analysis: lungs harvested at D2 from the mice of groups 5-6 were digested with collagenase and then filtered. Red blood cells were lysed using buffer and leukocytes were stained with the following antibodies: anti-CD45 (APC), anti-CD11b (VioGreen), anti-CD11c (VioBlue), anti-Siglec F (FITC), anti-CD64 (PE), anti-CD206 (PE Cy7), anti-I-Ab (PerCp Cy5.5), anti-Ly6C (AF700), anti-CD103 (APC Cy7), anti-Ly6G (BV711), anti-PDAC1 (BV605), anti-CD3 (FITC), anti-CD4 (VioGreen), anti-CD8 (PE), and anti-NK1.1 (PE Vio770). Cells were then fixed with PFA (paraformaldehyde) 4% before analysis with BD FACSAria Fusion device. The following cells populations were distinguished:

alveolar macrophages, interstitial macrophages, neutrophiles and recruited monocytes: [0319] alveolar macrophages: Siglec F.sup.+ CD11b.sup.−/low /CD64.sup.+CD11c.sup.+ [0320] interstitial macrophages: CD.sup.11b.sup.+ I-Ab.sup.+ CD64.sup.int/high [0321] neutrophils: CD11b.sup.+Ly6G.sup.+ [0322] monocyte derived macrophages: CD11b.sup.+ I-Ab.sup.−/low CD64.sup.+ Ly6C.sup.+/−
T cell subsets and NK cells: [0323] CD4.sup.+ T cells: CD3.sup.+NK1.1.sup.−CD4.sup.+CD8.sup.− [0324] CD8.sup.+ T cells: CD3.sup.+NK1.1.sup.−CD4.sup.−CD8.sup.+ [0325] NK cells: CD3.sup.−NK1.1.sup.+CD4.sup.−CD8.sup.−
dendritic cell subsets and plasmacytoid cells: [0326] CD11b+ dendritic cells: CD11b.sup.+ CD11c.sup.+/− I-Ab.sup.+ CD64.sup.− [0327] monocyte derived dendritic cells: CD11b.sup.+ I-Ab.sup.+ Ly6C.sup.+ [0328] CD103+ dendritic cells: CD103.sup.+ CD11c.sup.+ I-Ab.sup.+ [0329] conventional dendritic cells: CD11c.sup.+ CD11b.sup.−I-Ab.sup.+ [0330] plasmacytoid dendritic cells: CD11c.sup.+ PDCA1.sup.+I-Ab.sup.+

[0331] Cytokine measurement: cytokine levels were assessed in the blood samples collected on D1, D2, D3 and D4 from the mice of groups 1-4 (at D1 and D3 for groups 1 and 2, and at D2 and D4 for groups 3 and 4). sTREM1 was assessed by ELISA (mouse TREM-1 Quantikine ELISA kit, R&D Systems, R&D Systems, Bio-Techne). IL-10 (interleukin-10), KC (keratinocytes-derived chemokine also known as chemokine (C—X—C motif) ligand 1 or CXCL1), IL-6 (interleukin-6), IFNγ (interferon γ), IFNα (interferon α), IL-12p70 (interleukin-12 p70), MCP (monocyte chemoattractant protein-1 also known as chemokine ligand 2 or CCL2), RANTES (Regulated on Activation, Normal T cell Expressed and Secreted), IP-10 (interferon gamma-induced protein 10), and GM-CSF (granulocyte-macrophage colony-stimulating factor) were assessed with LEGENDplex™ Mouse Anti-Virus Response Panel (13-plex) with V-bottom Plate (Biolegend).

Results

[0332] Mice from groups 1 to 4 were followed on a daily basis from D0 to D10 for survival. Results are reported in FIG. 1. A significant reduction in mortality was observed in LR12-treated mice as compared to vehicle-treated mice, thus confirming that LR12 displayed a protective effect against mortality in mice infected with SARS-CoV-2.

[0333] The body weight of each mouse from groups 1 to 4 was monitored on a daily basis from D0 to D10. Data from groups 1 and 3 (vehicle-treated mice) were pooled. Data from groups 2 and 4 (LR12-treated mice) were pooled. Bodyweight assessments are reported in FIG. 2. The percentage of bodyweight loss from day 0 to day 10 was calculated for each mouse and reported in FIG. 3. The last observation carried forward (LOCF) approach was used to impute missing data due to death of animals. The body weight of mice infected with SARS-CoV-2 decreased from D1 to D10, whether they were treated with LR12 or vehicle. However, the administration of LR12 (white squares) resulted in a significant reduced bodyweight loss compared to vehicle (black circles). This suggests that LR12 had a protective effect against the infection with SARS-CoV-2.

Mice were also monitored for welfare and behavior. The evolution of the clinical score over time showed an apparition of first clinical signs at day 2 and 3 in all groups, and a worsening up to day 10. However, the administration of LR12 resulted in less severe alterations of mice welfare as compared to vehicle, with a mean clinical score of 3.5 in the LR12-treated group and 5 in the vehicle-treated group (FIG. 4). The assessment of the respiratory function confirmed this protective effect of LR12. Indeed, the monitoring of the respiratory rate or capacity of all mice showed that signs of decreased respiratory capacity were observed at day 2 in all groups, and a worsening up to day 10 (FIG. 5). However, the administration of LR12 resulted in a significant less severe alteration of mice respiration as compared to vehicle, with a mean respiratory score of 3 in the LR12-treated group and 5 in the vehicle group. This confirms that LR12 displayed a strong protective effect against the infection with SARS-CoV-2 on both the evolution of clinical signs and respiratory function.

[0334] Soluble TREM-1 (sTREM-1) is a marker of the activation of the TREM-1 pathway. Measuring sTREM-1 thus reflects the level of activation of the receptor. sTREM-1 is therefore a useful marker for the monitoring of treatment effect with a TREM-1 inhibitor. Plasma sTREM-1 concentrations were measured at 24, 48, 72, and 96 hours following the infection, and data are represented in FIG. 6. Results show a time-dependent increase of sTREM-1 over time, peaking at 72 hours following the infection in both LR12-treated and vehicle-treated groups. However, LR12-treated mice showed a decrease in sTREM-1 levels at 72 hours post-infection from 286.1 pg/mL (median of vehicle-treated group) to 132.3 pg/mL (median of LR12-treated group), p=0.0803. This confirms that LR12 administration was associated with an inhibition of TREM-1 receptor activation.

[0335] The evolution of the plasma concentration of several cytokines/chemokines: IFNγ (FIG. 7A), KC (FIG. 7B), MCP-1 (FIG. 7C), IL-12p70 (FIG. 7D), RANTES (FIG. 7E), IP-10 (CXCL10) (FIG. 7F), IL-10 (FIG. 7G), IFNα (FIG. 7H), IL-6 (FIG. 7I), and GM-CSF (FIG. 7J) was also assessed. Among these inflammatory markers, only IP-10, IL-10, IFNα, and IL-6 were significantly increased as compared to baseline values. Comparison of LR12-treated and vehicle-treated groups showed that LR12 was associated with a decrease in IP-10 and IFNα at all timepoints, a decrease of IL-6 at 72 hours, and an increase of IL-10 at 24 and 48 hours. This confirms that LR12 administration was associated with an immunomodulatory effect. IP-10 plays a role in the recruitment of several inflammatory cells such as monocytes/macrophages, neutrophils, T cells, NK cells and dendritic cells. IL-6 is a pleiotropic pro-inflammatory mediator, and high levels of IL-6 have been shown to be associated with severe forms of COVID-19. IL-10 is an anti-inflammatory mediator.

To further evaluate the effect of LR12 on the development of an immune response against SARS-Cov-2, the levels of various inflammatory cell-types were assessed in the lungs 48 hours post infection in LR-12-treated and vehicle-treated groups (FIGS. 8A-O). The total number of cells did not differ between both groups. A decrease in CD45.sup.+ cells was observed in the lungs of LR12-treated mice (FIG. 8A), which may be explained by a decrease in neutrophils and monocytes-derived macrophages. Indeed, the number of neutrophils (FIG. 8F) and monocytes-derived macrophages (FIG. 8C) in the lungs of LR12-treated mice were lower. LR12 had no effect on NK cells (FIG. 8K), CD8.sup.+ T cells (FIG. 8L), and CD4.sup.+ T cells (FIG. 8M). A lower number of NKT cells was also observed in the lungs of LR-12 treated mice (FIG. 8N). Although NKT cells contribute to the amplification of the antiviral immune response, these cells can also display increased expression of complement receptors and increased cytokine production, resulting in detrimental roles of NKT notably in the cytokine storm induction.
All these results confirm that TREM-1 inhibition with LR12 was associated with an immunomodulatory effect associated with a decreased inflammatory infiltrate, a decrease in the release of inflammatory mediators, which translated into a decrease in SARS-CoV-2 alteration of respiratory function and vital signs.

Example 2: Clinical Trial

[0336] A randomized, double-blind, placebo-controlled study is described herein, aiming at evaluating the safety, tolerability and efficacy of nangibotide (TLT-1 peptide having an amino acid sequence as set forth in SEQ ID NO: 10, also known as LR12) in mechanically ventilated patients suffering from COVID-19.

[0337] The study is a randomized, double-blind, placebo-controlled, in which one dose of nangibotide will be tested versus placebo.

Patient Randomization

[0338] A total of up to 730 patients suffering from COVID-19 and under mechanical ventilation (MV) are to be included in the study (including the 60 patients initially recruited). Randomization of patients is to be done in two stages to nangibotide or placebo. In stage one, 20 patients are randomized in a 1:1 ratio, in stage two, 40 patients are randomized in a 3:1 ratio to one of two treatment arms. The additional patients to be recruited will be randomized to nangibotide or placebo in a 1:1 ratio.

Treatment

[0339] Patients receive a continuous intravenous (i.v.) infusion of nangibotide at 1.0 mg/kg/h or a matching placebo. Treatment with study drug must be initiated as early as possible but no later than 48 hours after the initiation of invasive mechanical ventilation. Patients are treated for 5 days or until discharge from critical care (i.e., intensive care unit), whichever is sooner. The treatment with study drug is in addition to standard of care. The duration of the study is 28 days. A follow-up visit is performed on day 8 and day 14. The end of study visit is at day 28. A further follow up visit will be undertaken on day 60.

Inclusion Criteria

[0340] To be eligible for the study, patients must meet the following criteria: [0341] Provided informed consent (emergency consent according to local regulations where approved); [0342] Age 18 to 75 years (inclusive); [0343] Invasive mechanical ventilation (respiratory support using a mechanical ventilator delivered via an endotracheal tube or tracheostomy) for acute respiratory failure caused by COVID-19 for less than 48 hours; [0344] A PaO.sub.2/FiO.sub.2 ratio of <200 mmHg (<26.7 kPa); and [0345] Confirmed laboratory diagnosis of COVID-19 within 7 days of meeting screening criteria.

Exclusion Criteria

[0346] The presence of any of the following criteria excludes a patient from study enrolment: [0347] Known pregnancy (positive urine or serum pregnancy test); [0348] Currently receiving an immunomodulatory agent for the treatment of COVID-19 (including participation in clinical trials of such agents where treatment allocation is blinded or allocated on an open label basis); [0349] Weight>95 kg; [0350] Anticipated transfer to another hospital, which is not a study site within 72 hours; [0351] Expected to die within 6 months of treatment due to underlying chronic disease; or [0352] Limitations of care in place during current hospital admission.

Criteria for Evaluation

Primary Endpoint

[0353] The primary endpoint is the incidence of adverse events and mortality until day 28 and/or the clinical status (determined using the 7-point ordinal scale detailed below) assessed at day 28.

Secondary Endpoints

[0354] Additional Safety Parameters: [0355] Safety laboratory tests (as part of routine clinical care): hematology, coagulation, plasma biochemistry [0356] Adverse events (AEs), serious adverse events (SAEs) and deaths [0357] Suspected adverse drug reactions (serious and non-serious)

[0358] Efficacy Parameters: [0359] Improvement of clinical status until end of study using a 7 ordinal scale as detailed below on all study days until day 14, day 28 and day 60: [0360] 1—Not hospitalized, no limitations on activities [0361] 2—Not hospitalized, limitation on activities; [0362] 3—Hospitalized, not requiring supplemental oxygen; [0363] 4—Hospitalized, requiring supplemental oxygen; [0364] 5—Hospitalized, on non-invasive ventilation or high flow oxygen devices; [0365] 6—Hospitalized, on invasive mechanical ventilation or ECMO; [0366] 7—Death. [0367] Mortality at day 28 [0368] PaO.sub.2/FiO.sub.2 ratio [0369] Duration and nature of mechanical ventilation [0370] Incidence of thromboembolic events [0371] Incidence of secondary infections [0372] Duration and nature of other organ support therapies [0373] Functional status and mortality at day 60

[0374] Pharmacodynamics (exploratory):

sTREM-1, inflammatory exploratory biomarkers

Statistical Methods

Randomization and Stratification

[0375] In phase one, eligible patients are randomized in a 1:1 ratio of placebo or nangibotide into one of the two treatment arms. In phase two, eligible patients are randomized in a 1:3 ratio of placebo or nangibotide into one of the two treatment arms. The additional patients to be recruited will be randomized to nangibotide or placebo in a 1:1 ratio.

Sample Size Determination

[0376] The sample size of this safety study has not been based on a formal sample size calculation. The initial sample size of 60 patients (20 treated with placebo, 40 treated with nangibotide) should support the identification of the most frequent adverse effects of nangibotide in this patient population. Additional patients, up to a total number of 730 (including the initial 60 patients) are to be recruited.

Statistical Analyses

[0377] Primary Endpoint:

The primary endpoint is the incidence of adverse events and mortality until day 28. Usual descriptive statistics are to be used to analyze the safety parameters as follows: [0378] Adverse events (AEs), serious adverse events (SAEs) and death [0379] Safety laboratory tests: hematology, coagulation, plasma biochemistry

[0380] Secondary Endpoints:

All-Cause Mortality at Day 28

[0381] The difference in death rates at day 28 is to be estimated along with an asymptotic and exact 95% confidence interval. In addition, the all-cause mortality at day 28 is to be analyzed in an exact logistic regression model adjusting for treatment and categorized baseline sTREM-1 level.

Overall Survival

[0382] The Kaplan-Meier (KM) survival curves is to be provided with their 95% CI (confidence interval) for each group. A log-rank test is to be used to compare the treatment arms. In addition, a Proportional Hazard Cox model adjusting for treatment and categorized baseline sTREM-1 level is to be fitted to estimate the treatment effect expressed in terms of a hazard ratio with the 95% CI and p-value.

Clinical Status

[0383] The clinical status is a 7-point ordinal scale which will be assessed at baseline (day 1) until day 14 and on day 28. A descriptive analysis of each category is to be performed by treatment group.
Distribution of the 7-point ordinal scale is to be compared between groups with a Cochran-Mantel Haenszel test using modified ridit scores.