COMBINATION OF INHALED ANTIBODIES AND IMMUNOMODULATORY AGENTS FOR THE TREATMENT OR PREVENTION OF RESPIRATORY INFECTIONS

Abstract

A combination of agents capable of binding an infectious agent, such as an antibody, an antibody derivative, or an antibody mimetic, and immunomodulatory agents, such as probiotic strains, for the treatment or prevention of respiratory infections, in particular bacterial respiratory infections. The agents capable of binding an infectious agent are administered by inhalation.

Claims

1-15. (canceled)

16. A method of treating or preventing a respiratory infection in a subject, wherein the method comprises administering at least one agent capable of binding an infectious agent and at least one immunomodulatory agent to the subject, wherein the at least one agent capable of binding the infectious agent is administered by inhalation.

17. The method according to claim 16, wherein the at least one agent capable of binding the infectious agent is selected from the group consisting of an antibody, an antibody derivative, and an antibody mimetic.

18. The method according to claim 16, wherein the at least one immunomodulatory agent is selected from the group consisting of a probiotic strain, a mixture of probiotic strains, a Toll-like receptor agonist, a NOD-like receptor agonist, a RIG-like receptor agonist, a cytokine or mixture of cytokines, a chemokine or mixture of chemokines, an adjuvant, a flagellin, a flagellin variant, a polypeptide comprising one or more flagellin fragment(s), a CpG oligodeoxynucleotide (CpG ODN), α-galactosylceramide (α-Gal-Cer), aluminum salts, MF59, AS03, polyinosinic-polycytidylic acid, a polyphosphazene, an antibody directed against immune checkpoints, and mixtures thereof.

19. The method according to claim 16, wherein the at least one immunomodulatory agent is or comprises a probiotic strain or a mixture of probiotic strains selected from the Lactobacillaceae family

20. The method according to claim 19, wherein the at least one immunomodulatory agent is or comprises a probiotic strain or a mixture of probiotic strains selected from the species Lactobacillus murinus.

21. The method according to claim 20, wherein the at least one immunomodulatory agent is or comprises a probiotic strain selected from the strains deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on Apr. 14, 2015 under numbers CNCM I-4967 and CNCM I-4968, or the strain deposited on Apr. 16, 2018 under number CNCM I-5314, or a mixture thereof.

22. The method according to claim 16, wherein the infectious agent is selected from the group comprising or consisting of viruses, bacteria, fungi and parasites.

23. The method according to claim 22, wherein the infectious agent is a bacterium.

24. The method according to claim 23, wherein the infectious agent is a bacterium from the group ESKAPE.

25. The method according to claim 16, wherein the at least one agent capable of binding the infectious agent is directed against a molecule present on the surface of a bacterium.

26. The method according to claim 25, wherein the at least one agent capable of binding the infectious agent is directed against a molecule present on the surface of Pseudomonas aeruginosa.

27. The method according to claim 16, wherein said respiratory infection is an acute respiratory infection.

28. The method according to claim 27, wherein said respiratory infection is an acute lower respiratory tract infection.

29. The method according to claim 28, wherein said respiratory infection is selected from bronchitis, bronchiolitis, pneumonia, nosocomial pneumopathy, community-acquired pneumopathy, ventilator-assisted pneumopathy, influenza, or pertussis.

30. The method according to claim 16, wherein the subject suffers from a chronic respiratory pathology.

31. The method according to claim 30, wherein the chronic respiratory pathology is selected from the group consisting of chronic obstructive pulmonary disease (COPD), pulmonary interstitial diseases, lung cancer, adult asthma and pediatric asthma, bronchiectasis, rare and orphan lung diseases, cystic fibrosis and pulmonary vascular diseases.

32. The method according to claim 16, wherein the at least one agent capable of binding the infectious agent and the at least one immunomodulatory agent are administered separately in time.

33. The method according to claim 16, wherein the at least one agent capable of binding the infectious agent and the at least one immunomodulatory agent are administered simultaneously.

34. A method of treating or preventing a respiratory infection in a subject, wherein the method comprises administering a composition comprising or consisting essentially of a combination of at least one agent capable of binding an infectious agent and at least one immunomodulatory agent, wherein the at least one agent capable of binding the infectious agent and the at least one immunomodulatory agent are administered by inhalation.

35. The method according to claim 34, wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0332] FIG. 1 is a combination of drawings showing the experimental protocol for prophylactic treatment with antibodies and/or antibodies and probiotics. FIG. 1A: C57BL6/jRj mice (male, 7 weeks old), receive 50 μg of mAb166 via an intratracheal administration (Microsprayer®). 2 hours later, they are infected with Pseudomonas aeruginosa strain PA103, at a dose of 5×10.sup.5 cfu (colony forming unit)/40 μL/mouse via intratracheal administration. Clinical follow-up (survival, weight loss, clinical signs) of the animals is performed for 1 month after this primary infection. At D+33, the surviving individuals are reinfected, without additional treatment. Clinical follow-up is performed over 7 days. FIG. 1B: A cocktail of CNCM-I-4967 and CNCM-I-4968 strains, at 1×10.sup.6 cfu for each strain, is administered to animals at D (day)-3, D-2, D-1 intranasally (40 μL/mouse), prior to antibody treatment and infection as described in A.

[0333] FIG. 2 is a graph showing the survival of individuals to primary infection and reinfection with PA103 after prophylactic treatment with mAb166. Survival of individuals treated, according to the protocol described in FIG. 1, with mAb166 at 50 μg or a probiotic cocktail, or mAb166 at 50 μg combined with a probiotic cocktail or untreated, was analyzed after primary infection and reinfection. Results are representative of a pool of 5 independent experiments (n=10-30). (mAb166-50 μg+probiotics or mAb166-50 μg vs untreated groups ***: p<0.001/mAb166-50 μg+probiotics vs mAb166-50 μg groups ##: p<0.01, with a log-rank test).

[0334] FIG. 3 is a graph showing the pharmacokinetic analysis of mAb166 after prophylactic administration. The dosage of total mAb166 in serum and bronchoalveolar lavage (representative of the lower airways) was performed by ELISA at 1, 3, 7, 14, 21, and 28 days after antibody administration and PA103 infection. Data are expressed as mean±SEM (standard error of the mean). Results are representative of a pool of 3-5 independent experiments (n=5-30/analysis point). The dashed line indicates the limit of detection (ldd) of the assay.

[0335] FIG. 4 is a combination of drawings showing the experimental protocol of therapeutic treatment with antibody and/or antibody and probiotics. FIG. 4A: C57BL6/jRj mice (male, 7 weeks old) are infected with Pseudomonas aeruginosa strain PA103 at a dose of 3×10.sup.5ufc/40 μL/mouse via intratracheal administration and 1 hour later receive 50 μg of mAb166 via intratracheal administration (Microsprayer®). A clinical follow-up (survival, weight loss, clinical signs) of the animals is carried out during 1 month after this primary infection. At D+33, the surviving animals are reinfected, without additional treatment. Clinical follow-up is performed over 7 days. FIG. 4B: CNCM-I-4967, CNCM-I-4968, or CNCM-I-5314 at 1×1.sup.05 cfu for each strain, are administered to animals at D-3, D-2, D-1 intranasally (40 μL/mouse), prior to infection and antibody treatment as described in A.

[0336] FIG. 5 is a combination of graphs showing survival of individuals to primary infection and reinfection with PA103 after therapeutic treatment with mAb166. FIG. 5A: CNCM-I-4967 strain: Results are representative of a pool of 3 independent experiments (n=5-50). (mAb166-50 μg+4967 or mAb166-50 μg vs untreated groups *: p<0.05; ***: p<0.001/mAb166-50 μg+4967 vs mAb166-50 μg groups ###: p<0.001, with a log-rank test). FIG. 5B: CNCM-I-4968 strain: Results are representative of a pool of 3 independent experiments (n=5-30). (mAb166-50 μg+4967 or mAb166-50 μg vs untreated groups *: p<0.05; ***: p<0.001/mAb166-50 μg+4967 vs mAb166-50 μg groups ###: p<0.001, with a log-rank test). FIG. 5C: CNCM-I-5314 strain: Results are representative of a pool of 3 independent experiments (n=5-35). (mAb166-50 μg+4967 or mAb166-50 μg vs untreated groups *: p<0.05; ***: p<0.001/mAb166-50 μg+4967 vs mAb166-50 μg groups ###: p<0.001, with a log-rank test).

[0337] FIG. 6 is a graph showing the pharmacokinetic analysis of mAb166 after therapeutic administration. The dosage of total mAb166 in serum and bronchoalveolar lavage (representative of the lower airways) was performed by ELISA at 1, 3, 7, 14, 21, and 28 days after antibody administration and infection with PA103. Data are expressed as mean±SEM. Results are representative of a pool of 3-5 independent experiments (n=5-30/analysis point). The dotted line indicates the limit of detection (ldd) of the assay.

[0338] FIG. 7 is a graph showing the anti-PA103 humoral response in serum to reinfection with PA103 after therapeutic treatment with mAb166. The amount of PA103-specific immunoglobulin G in individuals treated, according to the protocol described in FIG. 4B, with mAb166 at 50 μg, or mAb166 at 50 μg combined with a probiotic strain or mAb166 at 50 μg combined with Flagellin (FLAMOD, 2.5 μg/mouse), was analyzed after reinfection by a semi-quantitative ELISA. Results are representative of a pool of 2 independent experiments (n=5-10). (mab166 vs combo groups *: p<0.05; **: p<0.01, with an Anova test). The dotted line indicates the limit of detection (ldd) of the assay.

EXAMPLES

[0339] The present invention will be better understood by reading the following example which illustrates the invention non-limitatively.

Example 1: Prophylactic treatment with antibodies and/or antibodies and probiotics

Materials and Methods

Experimental Protocol

[0340] In these experiments, the Pseudomonas aeruginosa strain PA103, was used as representative of strains involved in acute respiratory infections.

[0341] C57/BL6jRj mice (male, 7 weeks old) were infected once with PA103 with a pulmonary deposition of the bacterial inoculum (primary infection). Bacteria were diluted in PBS to a titer of 5.10.sup.5 bacteria/40 μL.

[0342] Two hours before, mice were administered by inhalation the mAb166 antibody (50 μg), a control IgG2b (MPC11 clone), or PBS solution via intratracheal administration using a MicroSprayer® aerosolizer (Penn-Century, USA) (FIG. 1A). mAb166 is a murine IgG2b specific for the perV protein, an essential component of the type 3 secretion system expressed on the surface of PA103.

[0343] For treatment of mice with probiotic strains, CNCM I-4967 and CNCM I-4968, derived from naïve mouse lungs and deposited on Apr. 14, 2015 at CNCM, were used. These strains were initially identified as belonging to Lactobacillus rhamnosus and Lactobacillus salivarius species, respectively, or genomically related species. Sequence analyses showed that these strains belong to the species Lactobacillus murinus.

[0344] Mice received a mixture of the probiotic strains CNCM I-4967 and 4968 (10.sup.6 cfu of each strain, 40 μL per mouse) by inhalation by intranasal route1, 2, and 3 days before priming (FIG. 1B).

[0345] After infection, survival and weight changes were monitored daily. In order to interrogate long-term protection induced by mAb166 antibody, animals surviving the primary infection were reinfected (secondary infection) at D+33 after the primary infection, without additional treatment. Their survival and the associated immune response are analyzed.

Pharmacokinetic Analysis of mAb166 after Prophylactic Administration

[0346] The determination of total mAb166 in serum and bronchoalveolar lavage (representative of the lower airways) was performed by ELISA at 1, 3, 7, 14, 21, and 28 days after antibody administration and infection with PA103.

Results

Short-Term Protection Induced by Inhaled Antibody against Pa (Pseudomonas aeruginosa) in Combination with Probiotic Strains

[0347] As shown in FIG. 2, the data reveal that animals treated with the anti-Pa mAb166 (50 μg) antibody administered by inhalation show a very significant improvement in survival compared to untreated animals (i.e., receiving the PBS solution) during primary infection. However, this protection is only partial, as the survival rate is about 70%. In addition, data show that administration of a control IgG2b (clone MPC11) has no positive impact on survival of treated animals during primary infection (data not shown).

[0348] These data also reveal that animals treated with a combination of the inhaled anti-Pa mAb166 antibody (50 μg) with a mixture of the probiotic strains CNCM I-4967 and CNCM-4968 show superior survival (greater than 90%) compared to untreated animals or animals treated with the anti-Pa mAb166 antibody alone during a first infection (FIG. 2). In contrast, administration of the probiotic strains CNCM I-4967 and CNCM-4968 alone does not affect survival.

[0349] Thus, the data indicate that the combination of an inhaled anti-Pa antibody with a mixture of probiotic strains is more effective in treating a first respiratory Pa infection.

Long-Term Protection Induced by Inhaled Anti-Pa Antibody in Combination with Probiotic Strains

[0350] Surviving animals were reinfected at D+33 after primary infection, when anti-Pa mAb166 antibody was no longer detectable in blood and airways (FIG. 3), with the same dose of Pa as during primary infection.

[0351] Animals treated with the anti-Pa mAb166 antibody alone showed a better survival (less than 40%) after reinfection (or second infection) compared to untreated animals (FIG. 2), showing that Pa-infected animals treated with an inhaled antibody show a memory response allowing them to control a reinfection (or second infection) (by the same pathogen) in the absence of additional treatment during reinfection.

[0352] Interestingly, animals treated with the combination of the anti-Pa mAb166 antibody and the mixture of probiotic strains showed a significantly higher survival (around 80%) than animals that were not treated or were treated with the anti-Pa mAb166 antibody alone (FIG. 2). Administration of the probiotic strains alone had no significant effect. These data indicate that the combination of an inhaled antibody with the mixture of probiotic strains is more effective than the antibody alone in controlling reinfection (or second infection) with the same pathogen.

[0353] Thus, these data demonstrate that administration of a combination of an inhaled anti-infective antibody with a mixture of probiotic strains at the time of primary infection improves the short-term and long-term efficacy of an anti-infective antibody.

Example 2: Therapeutic Treatment with Antibodies and/or Antibodies and Probiotics

Materials and Methods

Experimental Protocol

[0354] C57BL6/jRj mice (male, 7 weeks old) were infected with Pseudomonas aeruginosa strain PA103 at a dose of 3×10.sup.5 cfu/40 μL/mouse by inhalation via intratracheal administration and then received 1 hour later 50 μg of mAb166 via intratracheal administration (Microsprayer®) (FIG. 4A). Clinical follow-up (survival, weight loss, clinical signs) of the animals was performed for 1 month after this primary infection. At D+33, surviving individuals were reinfected, without additional treatment. Clinical follow-up was performed over 7 days.

[0355] For treatment of mice with probiotic strains, CNCM-I-4967, CNCM-I-4968, or CNCM-I-5314 at 1×10.sup.5 cfu for each strain, were administered to the animals at D-3, D-2, D-1 by inhalation by intranasal route (40 μL/mouse), prior to infection and antibody treatment as described in FIG. 4B.

Pharmacokinetic Analysis of mAb166 after Therapeutic Administration

[0356] The dosage of total mAb166 in serum and bronchoalveolar lavage (representative of the lower airway) was performed by ELISA at 1, 3, 7, 14, 21, and 28 days after PA103 infection and antibody administration.

Results

Short-Term Treatment Induced by Inhaled Antibody to Pa (Pseudomonas aeruginosa) in Combination with Probiotic Strains

[0357] The results in FIG. 5A-C show that animals treated with the antibody in combination with one of the strains CNCM I-4967, CNCM I-4968, or CNCM I-5314 had better survival compared with untreated animals or animals treated with the mAb166 antibody alone during a first infection. In contrast, administration of one of the probiotic strains alone did not positively affect survival.

[0358] These data show that the combination of an inhaled anti-Pa antibody with a probiotic strain is more effective in treating a first Pa infection.

Long-Term Treatment Induced by Inhaled Anti-Pa Antibody in Combination with Probiotic Strains

[0359] Surviving animals were reinfected at D+33 after primary infection, when mAb166 antibody was no longer detectable in blood and airways (FIG. 6), with the same dose of Pa as in the primary infection.

[0360] Animals treated with mAb166 antibody alone showed improved survival (about 30%) after reinfection (or second infection) compared to untreated animals (FIG. 5A-C), showing that Pa-infected animals treated with inhaled antibody have a memory response that allows them to control a second infection (with the same pathogen).

[0361] In contrast, animals treated with the combination of the mAb166 antibody and one of the CNCM I-4967, CNCM I-4968 or CNCM I-5314 strains showed a higher survival (between 60 and 80%) than animals that were not treated or were treated with the mAb166 antibody alone (FIG. 5A-C).

[0362] Thus, these data indicate that administration of a combination of an inhaled anti-Pa antibody with a probiotic strain at the time of primary infection can control both a first respiratory infection and also repeated respiratory infections when there are no inhaled anti-Pa antibodies or probiotic strains left in the body.

Example 3: Humoral Response Induced during Reinfection with P. aeruginosa Following Treatment During Primary Infection with Anti-Pa Antibody, Anti-Pa Antibody Combined with a Probiotic Strain and Anti-Pa Antibody Combined with Flagellin

Materials and Methods

[0363] The amount of PA103-specific immunoglobulin G in individuals treated, according to the protocol described in FIG. 4B, with mAb166 at 50 μg, or mAb166 at 50 μg combined with a probiotic strain or mAb166 at 50 μg combined with Flagellin (FLAMOD, 2.5 μg/mouse), was analyzed 5 days after reinfection by a semi-quantitative ELISA.

Results

[0364] The results in FIG. 7 indicate that upon reinfection, the combination of an inhaled anti-Pa mAb166 antibody with a probiotic strain such as CNCM I-4967 and CNCM I-4968 induces a greater amount of PA103-specific immunoglobulin G in the serum of treated animals than treatment with an anti-Pa mAb166 antibody alone.

[0365] Interestingly, upon reinfection, the combination of inhaled anti-Pa mAb166 antibody in combination with flagellin also induces more PA103-specific immunoglobulin G in the serum of treated animals than treatment with anti-Pa mAb166 antibody alone (FIG. 7). Of note, individuals treated with flagellin alone during primary infection were not resistant to infection.

[0366] These results demonstrate that the humoral response upon reinfection is better when an inhaled anti-infective antibody was used in combination with a probiotic strain or flagellin than treatment with an antibody alone at the time of primary infection, thus effectively preventing recurrent respiratory infections in the long term