Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
USE OF TIM-3 INHIBITORS FOR THE TREATMENT OF EXACERBATIONS IN PATIENTS SUFFERING FROM SEVERE ASTHMA
Document Type and Number:
WIPO Patent Application WO/2020/070062
Kind Code:
A1
Abstract:
Rhinovirus infections are the main cause of asthma exacerbations. As Natural Killer (NK) cells are important actors of the antiviral innate response, the inventors aimed to evaluate the functions of NK cells from severe asthmatic patients in response to rhinovirus or rhinovirus-like molecules. Peripheral blood mononuclear cells from severe asthmatic patients and healthy donors were stimulated with pathogen-like molecules or with the human rhinovirus A9 (HRV). NK cell activation, degranulation and IFN-γ expression were analyzed. The inventors found NK cells from severe asthmatic patients were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of Tim-3. Finally, the inventors show that neutralization of Tim3 restores rhinovirus-induced NK cell activation. Together, the findings prompt to consider that Tim-3 inhibitors would be suitable for treating exacerbation in patients suffering from severe asthma.

Inventors:
DUEZ CATHERINE (FR)
DEVULDER JUSTINE (FR)
CHENIVESSE CÉCILE (FR)
Application Number:
PCT/EP2019/076437
Publication Date:
April 09, 2020
Filing Date:
September 30, 2019
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
INST NAT SANTE RECH MED (FR)
UNIV LILLE (FR)
CENTRE NAT RECH SCIENT (FR)
PASTEUR INSTITUT (FR)
CENTRE HOSPITALIER REGIONAL UNIV LILLE (FR)
International Classes:
C07K16/28; A61K31/573; A61P11/06; G01N33/569
Domestic Patent References:
WO2017019897A12017-02-02
WO1993011161A11993-06-10
WO2006030220A12006-03-23
WO2006003388A22006-01-12
WO2011159877A22011-12-22
WO2008119353A12008-10-09
WO2011131746A22011-10-27
WO2011155607A12011-12-15
WO2013006490A22013-01-10
WO2010117057A12010-10-14
WO2015117002A12015-08-06
WO1996034103A11996-10-31
WO1994004678A11994-03-03
Foreign References:
EP0404097A21990-12-27
EP0368684A11990-05-16
US4816567A1989-03-28
US5225539A1993-07-06
US5585089A1996-12-17
US5693761A1997-12-02
US5693762A1997-12-02
US5859205A1999-01-12
US5591669A1997-01-07
US5598369A1997-01-28
US5545806A1996-08-13
US5545807A1996-08-13
US6150584A2000-11-21
US6194551B12001-02-27
US6566135B12003-05-20
US6566131B12003-05-20
US6365354B12002-04-02
US6410323B12002-06-25
US6107091A2000-08-22
US6046321A2000-04-04
US5981732A1999-11-09
US8697359B12014-04-15
US20140068797A12014-03-06
Other References:
JENNIFER KEARLEY ET AL: "Th-2 driven, allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 204, no. 6, 1 June 2007 (2007-06-01), ROCKEFELLER UNIVERSITY PRESS, US, pages 1289 - 1294, XP008133823, ISSN: 0022-1007, DOI: 10.1084/JEM.20062093
LI ZHENHONG ET AL: "The T-cell immunoglobulin and mucin domain (Tim) gene family in asthma, allergy, and autoimmunity", ALLERGY AND ASTHMA PROCEEDINGS, vol. 34, no. 1, 31 December 2012 (2012-12-31), OCEANSIDE PUBLICATIONS, INC, US, pages e21 - e26, XP009511367, ISSN: 1539-6304, DOI: 10.2500/AAP.2013.34.3646
LU XIAO-XIA ET AL: "Small interfering RNA targeting T-cell Ig mucin-3 decreases allergic airway inflammation and hyperresponsiveness.", INFLAMMATION, vol. 36, no. 3, June 2013 (2013-06-01), pages 582 - 591, XP002789188, ISSN: 1573-2576
LOMMATZSCH MAREK ET AL: "Severe asthma: definition, diagnosis and treatment.", DEUTSCHES ARZTEBLATT INTERNATIONAL, vol. 111, no. 50, 12 December 2014 (2014-12-12), pages 847 - 855, XP002789189, ISSN: 1866-0452
GORSKA MAGDALENA M: "Natural killer cells in asthma.", CURRENT OPINION IN ALLERGY AND CLINICAL IMMUNOLOGY FEB 2017, vol. 17, no. 1, February 2017 (2017-02-01), pages 50 - 54, XP055563197, ISSN: 1473-6322
DENNIS M ET AL: "EFFECTIVENESS AND COMPLICATIONS OF AEROSOL DEXAMETHASONE PHOSPHATE IN SEVERE ASTHMA.", THE JOURNAL OF ALLERGY, vol. 35, January 1964 (1964-01-01), pages 70 - 76, XP002789191, ISSN: 0021-8707
LU XIAO-XIA ET AL: "Dexamethasone reduces IL-17 and Tim-3 expression in BALF of asthmatic mice.", JOURNAL OF HUAZHONG UNIVERSITY OF SCIENCE AND TECHNOLOGY. MEDICAL SCIENCES = HUA ZHONG KE JI DA XUE XUE BAO. YI XUE YING DE WEN BAN = HUAZHONG KEJI DAXUE XUEBAO. YIXUE YINGDEWEN BAN3, vol. 33, no. 4, August 2013 (2013-08-01), pages 479 - 484, XP002789192, ISSN: 1672-0733
MORGAN DAVID J ET AL: "Distinct Effects of Dexamethasone on Human Natural Killer Cell Responses Dependent on Cytokines.", FRONTIERS IN IMMUNOLOGY, vol. 8, 432, 2017, pages 1 - 15, XP002789193, ISSN: 1664-3224, DOI: 10.3389/fimmu.2017.00432
BARLOW J L ET AL: "Tim1 and Tim3 are not essential for experimental allergic asthma.", CLINICAL AND EXPERIMENTAL ALLERGY : JOURNAL OF THE BRITISH SOCIETY FOR ALLERGY AND CLINICAL IMMUNOLOGY JUL 2011, vol. 41, no. 7, July 2011 (2011-07-01), pages 1012 - 1021, XP002796014, ISSN: 1365-2222
SABATOS ET AL., NAT IMMUNOL, vol. 4, no. 11, 2003, pages 1102
WARD ET AL., NATURE, vol. 341, no. 6242, 12 October 1989 (1989-10-12), pages 544 - 6
HOLT ET AL., TRENDS BIOTECHNOL., vol. 21, no. 11, 2003, pages 484 - 490
WU ET AL.: "Antibody Engineering, Springer Berlin Heidelberg", 2010, article "Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule"
ZETSCHE ET AL.: "Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System", CELL, vol. 163, 2015, pages 1 - 13
LAMBRECHT, B. N.HAMMAD, H.: "The immunology of asthma", NAT. IMMUNOL., vol. 16, 2014, pages 45 - 56
CHUNG, K. F. ET AL.: "International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma", EUR. RESPIR. J., vol. 43, 2014, pages 343 - 373
STONE, B. ET AL.: "Characterizing patients with asthma who received Global Initiative for Asthma steps 4-5 therapy and managed in a specialty care setting", ALLERGY ASTHMA PROC., vol. 39, 2018, pages 27 - 35
CHASTEK, B. ET AL.: "Economic Burden of Illness Among Patients with Severe Asthma in a Managed Care Setting", J. MANAG. CARE SPEC. PHARM., vol. 22, 2016, pages 848 - 861
DALAL, A. A. ET AL.: "Dose-Response Relationship Between Long-Term Systemic Corticosteroid Use and Related Complications in Patients with Severe Asthma", J. MANAG. CARE SPEC. PHARM., vol. 22, 2016, pages 833 - 847
MCDONALD, V. M.GIBSON, P. G.: "Exacerbations of severe asthma", CLIN. EXP. ALLERGY, vol. 42, 2012, pages 670 - 677
SYKES, A.JOHNSTON, S. L.: "Etiology of asthma exacerbations", J. ALLERGY CLIN. IMMUNOL., vol. 122, 2008, pages 685 - 688, XP025493905, doi:10.1016/j.jaci.2008.08.017
EDWARDS, M. R.BARTLETT, N. W.HUSSELL, T.OPENSHAW, P.JOHNSTON, S. L.: "The microbiology of asthma", NAT. REV. MICROBIOL., 2012
MESSAGE, S. D. ET AL.: "Rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and Thl/2 cytokine and IL-10 production", PROC. NATL. ACAD. SCI., vol. 105, 2008, pages 13562 - 13567
WARK, P. A. B.: "Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhino virus", J. EXP. MED., vol. 201, 2005, pages 937 - 947
CONTOLI, M. ET AL.: "Role of deficient type III interferon-^ production in asthma exacerbations", NAT. MED., vol. 12, 2006, pages 1023 - 1026, XP002414169, doi:10.1038/nm1462
LAZA-STANCA, V. ET AL.: "The Role ofIL-15 Deficiency in the Pathogenesis of Virus-Induced Asthma Exacerbations", PLOS PATHOG, vol. 7, 2011, pages e1002114
CHAIRAKAKI, A.-D. ET AL.: "Plasmacytoid dendritic cells drive acute exacerbations of asthma", J. ALLERGY CLIN. IMMUNOL., 2017
JACOBS, S. E.LAMSON, D. M.ST GEORGE, K.WALSH, T., J. HUMAN RHINOVIRUSES. CLIN. MICROBIOL. REV., vol. 26, 2013, pages 135 - 162
VIVIER, E.TOMASELLO, E.BARATIN, M.WALZER, T.UGOLINI, S.: "Functions of natural killer cells", NAT. IMMUNOL., vol. 9, 2008, pages 503 - 510, XP055316463, doi:10.1038/ni1582
LONG, E. O.SIK KIM, H.LIU, D.PETERSON, M. E.RAJAGOPALAN, S.: "Controlling Natural Killer Cell Responses: Integration of Signals for Activation and Inhibition", ANNU. REV. IMMUNOL., vol. 31, 2013, pages 227 - 258, XP055558564, doi:10.1146/annurev-immunol-020711-075005
MANDELBOIM, O. ET AL.: "Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells", NATURE, vol. 409, 2001, pages 1055 - 1060, XP002191494, doi:10.1038/35059110
ADIB-CONQUY, M.SCOTT-ALGARA, D.CAVAILLON, J.-M.SOUZA-FONSECA-GUIMARAES, F.: "TLR-mediated activation of NK cells and their role in bacterial/viral immune responses in mammals", IMMUNOL. CELL BIOL., vol. 92, 2014, pages 256 - 262
CALIGIURI, M. A.: "Human natural killer cells", BLOOD, vol. 112, 2008, pages 461 - 469, XP055159550, doi:10.1182/blood-2007-09-077438
BARNIG, C. ET AL.: "Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma", SCI. TRANSL. MED., vol. 5, 2013, pages 174ra26
DUVALL, M. G. ET AL.: "Natural killer cell-mediated inflammation resolution is disabled in severe asthma", SCI. IMMUNOL., vol. 2, 2017, pages eaam5446
LIN, S.-J. ET AL.: "Decreased intercellular adhesion molecule-1 (CD54) and L-selectin (CD62L) expression on peripheral blood natural killer cells in asthmatic children with acute exacerbation", ALLERGY, vol. 58, 2003, pages 67 - 71
AWAD, A. ET AL.: "Natural Killer Cells Induce Eosinophil Activation and Apoptosis", PLOS ONE, vol. 9, 2014, pages e94492
GHADIALLY, H. ET AL.: "NKp46 regulates allergic responses", EUR. J. IMMUNOL., vol. 43, 2013, pages 3006 - 3016
KORSGREN, M. ET AL.: "Natural Killer Cells Determine Development of Allergen-induced Eosinophilic Airway Inflammation in Mice", J. EXP. MED., vol. 189, 1999, pages 553 - 562
HASPESLAGH, E. ET AL.: "Role of NKp46 + natural killer cells in house dust mite - driven asthma", EMBO MOL. MED., 2018, pages e8657
HAWORTH, O.CERNADAS, M.LEVY, B. D.: "NK Cells Are Effectors for Resolvin El in the Timely Resolution of Allergic Airway Inflammation", J. IMMUNOL., vol. 186, 2011, pages 6129 - 6135, XP055171267, doi:10.4049/jimmunol.1004007
SIMONS, B. ET AL.: "PGI 2 Controls Pulmonary NK Cells That Prevent Airway Sensitization to House Dust Mite Allergen", J. IMMUNOL., vol. 198, 2017, pages 461 - 471
FERRINI, M. E. ET AL.: "CB2 receptors regulate natural killer cells that limit allergic airway inflammation in a murine model of asthma", ALLERGY, vol. 72, 2017, pages 937 - 947
LUNDING, L. P. ET AL.: "Poly(inosinic-cytidylic) Acid-Triggered Exacerbation of Experimental Asthma Depends on IL-17A Produced by NK Cells", J. IMMUNOL., vol. 194, 2015, pages 5615 - 5625
COOPER, G.KHAKOO, S.WILKINSON, T.STAPLES, K. LSC: "Tissue-resident Natural Killer (NK) cell Phenotype in the Human Lung", PA2013 (EUROPEAN RESPIRATORY SOCIETY, 2017, 2017
SCORDAMAGLIA, F. ET AL.: "Perturbations of natural killer cell regulatory functions in respiratory allergic diseases", J. ALLERGY CLIN. IMMUNOL., vol. 121, 2008, pages 479 - 485, XP022471336, doi:10.1016/j.jaci.2007.09.047
CIBRIAN, D.SANCHEZ-MADRID, F.: "D69: from activation marker to metabolic gatekeeper", EUR J IMMUNOL., vol. 47, no. 6, 2017, pages 946 - 953
KENNEDY, J. L. ET AL.: "Comparison of Viral Load in Individuals with and without Asthma during Infections with Rhino virus", AM. J. RESPIR. CRIT. CARE MED., vol. 189, 2014, pages 532 - 539
HOSOKI, K. ET AL.: "Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IL-8 Levels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma, and Correlates Inversely with FEV1", PLOS ONE, vol. 10, no. 5, 2015, pages e0126035
TAN, K.S.: "In Vitro Model of Fully Differentiated Human Nasal Epithelial Cells Infected With Rhinovirus Reveals Epithelium-Initiated Immune Responses", J INFECT DIS., vol. 217, no. 6, 2018, pages 906 - 915
LUCI, C. ET AL.: "Peripheral natural killer cells exhibit qualitative and quantitative changes in patients with psoriasis and atopic dermatitis", BR. J. DERMATOL., vol. 166, 2012, pages 789 - 796
GILL, M. A. ET AL.: "Counterregulation between the Fc RI Pathway and Antiviral Responses in Human Plasmacytoid Dendritic Cells", J. IMMUNOL., vol. 184, 2010, pages 5999 - 6006
RUPANI, H. ET AL.: "Toll-like Receptor 7 Is Reduced in Severe Asthma and Linked to an Altered MicroRNA Profile", AM. J. RESPIR. CRIT. CARE MED., vol. 194, 2016, pages 26 - 37
PARSONS, K. S.HSU, A. C.WARK, P. A. B.: "TLR3 and MDA5 signalling, although not expression, is impaired in asthmatic epithelial cells in response to rhinovirus infection", CLIN. EXP. ALLERGY J. BR. SOC. ALLERGY CLIN. IMMUNOL., vol. 44, 2014, pages 91 - 101
LIMA, J. F.OLIVEIRA, L. M. S.PEREIRA, N. Z.DUARTE, A. J. S.SATO, M. N.: "Polyfunctional natural killer cells with a low activation profile in response to Toll-like receptor 3 activation in HIV-1-exposed seronegative subjects", SCI. REP., vol. 7, 2017, pages 524
Attorney, Agent or Firm:
INSERM TRANSFERT (FR)
Download PDF:
Claims:
CLAIMS:

1. A method of enhancing the cytotoxic activity of NK cells in a patient suffering from severe asthma comprising administering to the subject a therapeutically effective amount of a Tim-3 inhibitor. 2. A method of reducing NK cell exhaustion in a patient suffering from severe asthma comprising administering to the subject a therapeutically effective amount a Tim-3 inhibitor.

3. A method of therapy in a patient suffering from severe asthma, comprising administering to the patient a therapeutically effective amount of a Tim-3 inhibitor, wherein said administration enhances the cytotoxic activity of NK cells in the patient.

4. The method of claim 3 for the treatment of exacerbation in a patient suffering from severe asthma.

5. The method of claim 4 wherein the exacerbation results from a human rhino virus (HRV) infection.

6. The method of claim 1, 2 or 3 wherein the Tim-3 inhibitor is an antibody having specificity for Tim-3.

7. The method of claim 6 wherein the antibody comprises:

(a) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 2 (GYTFTSY); a VHCDR2 amino acid sequence of SEQ ID NO: 3 (YPGNGD); and a VHCDR3 amino acid sequence of SEQ ID NO: 4

(VGGAFPMDY); and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 5 (SESVEYYGTSL), a VLCDR2 amino acid sequence of SEQ ID NO: 6 (AAS), and a VLCDR3 amino acid sequence of SEQ ID NO: 7 (SRKDPS); - (b) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 8

(SYNMH); a VHCDR2 amino acid sequence of SEQ ID NO: 9 (DIYPGNGDTSYNQKFKG); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 10 (RASES VEYYGTSLMQ), a VLCDR2 amino acid sequence of SEQ ID NO: 11 (AASNVES), and a VLCDR3 amino acid sequence of SEQ ID NO: 12 (QQSRKDPST);

(c) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 2 (GYTFTSY); a VHCDR2 amino acid sequence of SEQ ID NO: 13 (YPGSGD);; and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (V GGAFPMD Y); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 5 (SESVEYYGTSL), a VLCDR2 amino acid sequence of SEQ ID NO: 6 (AAS), and a VLCDR3 amino acid sequence of SEQ ID NO: 7 (SRKDPS);

(d) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 8 (SYNMH); a VHCDR2 amino acid sequence of SEQ ID NO: 14 (DIYPGSGDTSYNQKFKG); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 10 (RASESVEYYGTSLMQ), a VLCDR2 amino acid sequence of SEQ ID NO: 11 (AASNVES), and a VLCDR3 amino acid sequence of SEQ ID NO: 12 (QQSRKDPST);

(e) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 2 (GYTFTSY); a VHCDR2 amino acid sequence of SEQ ID NO: 15 (YPGQGD); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 5 (SESVEYYGTSL), a VLCDR2 amino acid sequence of SEQ ID NO: 6 (AAS), and a VLCDR3 amino acid sequence of SEQ ID NO: 7 (SRKDPS); or

(f) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 8 (SYNMH); a VHCDR2 amino acid sequence of SEQ ID NO: 16 (DIYPGQGDTSYNQKFKG); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 10 (RASESVEYYGTSLMQ), a VLCDR2 amino acid sequence of SEQ ID NO: 11 (AASNVES), and a VLCDR3 amino acid sequence of SEQ ID NO: 12 (QQSRKDPST).

8. The method of claim 1, 2 or 3 wherein the Tim-3 inhibitor comprise all or a portion of the extracellular domains of Tim-3 so as to form a soluble receptor that is capable to trap a Tim-3 ligand.

9. The method of claim 1, 2 or 3 wherein the Tim-3 inhibitor is an inhibitor of Tim-3 expression, such as siR A or an antisense oligonucleotide.

Description:
USE OF TIM-3 INHIBITORS FOR THE TREATMENT OF EXACERBATIONS IN PATIENTS SUFFERING FROM SEVERE ASTHMA

FIELD OF THE INVENTION:

The present invention relate to methods of treating exacerbations in patients suffering from severe asthma.

BACKGROUND OF THE INVENTION:

Asthma is a chronic inflammatory disease characterized by reversible airway obstruction, inflammation of the airways, remodelling of the lung tissue and airway hyperresponsiveness 1 . Worldwide, 300 million people have asthma, with 5 to 10 percent suffering from severe asthma. According to the European Respiratory Society and American Thoracic Society, severe asthma is asthma which requires treatment with high doses of corticosteroid and p 2 -adrenergic receptor agonist to prevent it from becoming uncontrolled or which remains uncontrolled despite therapy 2 . Severe asthmatic patients experience poor quality of life, a higher number of hospitalizations and a higher risk of mortality 3-5 . Severe asthmatic patients are prone to frequent exacerbations. Asthma exacerbation is defined as the deterioration in the patient’s symptoms requiring the use of systemic corticosteroids and/or hospitalization to prevent a serious outcome 6 . Approximately 50 to 80% of acute asthma exacerbations are associated with respiratory viral infections (in adults and children respectively) and, of viruses implicated, 50-60% are human rhino viruses (HRV) 7,8 . Mechanisms underlying asthma exacerbation remain poorly understood, however, it is clear that people with asthma have increased susceptibility to lower respiratory virus infection 9 . Diminished production of type I and III IFN by bronchial epithelial cells from asthmatic patients has been reported 10,11 . The induction of IL-15 by HRV in alveolar macrophages from asthmatic subjects is deficient in vitro, and is correlated to airway hyperresponsiveness and the severity of symptoms 12 . In a mouse model of rhinovirus-induced exacerbation of asthma, depletion of plasmacytoid dendritic cells during the viral infection abrogate the inflammation burden 13 . All together, these results suggest that the innate antiviral immunity may be defective in asthmatic patients. Most rhino viruses initiate the innate antiviral response by adhering to ICAM-l on bronchial epithelial cells, leading to the endocytosis of the viruses into the cells. HRV activate the innate immune system through pattern recognition receptor (PRR): the capside is recognized by Toll-Like Receptor (TLR)2, single strand RNA and double strand RNA from rhinoviruses are recognized by TLR3, TLR7/8, retinoic acid-inducible gene (RIG)-l and melanoma differentiation- associated protein (MDA)-5. Engagement of these receptors induces cytokine expression including type I and type III interferons, but also IL-6, IL-12, and IL-15 14 .

Natural Killer (NK) cells are important actors of the antiviral innate immune response 15 . Their activation depends on the integration of multiple signals from innate receptors. Some receptors can recognize major histocompatibility complex class (MHC)-I and inhibit NK cell activation, whereas others can recognize cytokines, chemokines, and microbial components leading to the activation of NK cells 16 . NKp46 is an activating receptor of the Natural Cytotoxic Receptor (NCR) family. To date, few pathogen-associated ligands and cellular co-ligands for NKp46 have been documented. Influenza hemagglutinin protein and hemagglutinin- neuraminidase of parainfluenza virus can bind to NKp46 and induce NK activation 17 . NK cells also express several PRR: TLR2, TLR3, TLR4, TLR7/8, TLR9, NOD2 (nucleotide-binding oligomerization domain 2), NLRP3, RIG-I. TLR were shown to activate NK cell functions either directly or in cooperation with accessory cells in a cytokine or cell-to-cell contact- dependent manner 18 . Human NK cells are classically identified by their surface expression of CD56 and a lack of CD3, and can be categorized into two, developmentally related but functionally distinct, subsets based on relative CD56 expression: CD56 bnght and CD56 dim NK cells 19 .

Although modifications in human NK cell phenotype and functional capacities have been observed in samples from asthmatic patients 20,21 , including after acute exacerbation 22 , the role of NK cells in the asthmatic disease remains unclear. NK cells from healthy individuals may regulate eosinophilic inflammation by inducing eosinophil activation and apoptosis 23 . However, peripheral blood NK cells from severe asthmatic have reduced capacity to induce eosinophil apoptosis, despite increased activation status 20 . Mouse models of allergic asthma highlight a complex role of NK cells, which may participate 24,25 or not 26 to allergic inflammation, or may resolve it 27-29 . In a mouse model of asthma exacerbation with a TLR3 agonist, NK cells express IL-17A and may increase the lung inflammation 30 .

SUMMARY OF THE INVENTION:

The present invention relate to methods of treating exacerbations in patients suffering from severe asthma. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Rhinovirus infections are the main cause of asthma exacerbations. As Natural Killer (NK) cells are important actors of the antiviral innate response, the inventors aimed to evaluate the functions of NK cells from severe asthmatic patients in response to rhinovirus or rhinovirus like molecules. Peripheral blood mononuclear cells from severe asthmatic patients and healthy donors were stimulated with pathogen-like molecules or with the human rhinovirus A9 (HRV). NK cell activation, degranulation and IFN-g expression were analyzed. The inventors found NK cells from severe asthmatic patients to be less cytotoxic in response to TLR3 or TLR7/8 stimulation and to HRV. After culture with IL-12+IL-15, cytokines which are produced during viral infections, NK cells from severe asthmatic patients were less cytotoxic and expressed less IFN-g than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of Tim-3. Finally, the inventors show that neutralization of Tim3 restores rhinovirus-induced NK cell activation. Together, the findings indicate that the activation of NK cells may be insufficient during respiratory infections in severe asthmatic patients and prompt to consider that Tim-3 inhibitors would be suitable for treating exacerbation in patients suffering from severe asthma.

Accordingly, the first object of the present invention relates to a method of enhancing the cytotoxic activity of NK cells in a patient suffering from severe asthma comprising administering to the subject a therapeutically effective amount of a Tim-3 inhibitor.

More specifically, the present invention provides a method of therapy in a patient suffering from severe asthma, comprising administering to the patient a therapeutically effective amount of a Tim-3 inhibitor, wherein said administration enhances the cytotoxic activity of NK cells in the patient.

More particularly, the present invention provides a method of reducing NK cell exhaustion in a patient suffering from severe asthma comprising administering to the subject a therapeutically effective amount a Tim-3 inhibitor.

As used herein, the term "NK cell" has its general meaning in the art and refers to a sub population of lymphocytes that are involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD 16, CD56 and/or CD57, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express“self’ MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. The ability of the Tim-3 inhibitor to enhance cytotoxic activity of NK cells may be determined by any assay well known in the art. Typically said assay is an in vitro assay wherein NK cells are brought into contact with target cells (e.g. target cells that are recognized and/or lysed by NK cells). For example, the Tim-3 inhibitor can be selected for the ability to increase specific lysis by NK cells by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%, or more of the specific lysis obtained at the same effector: target cell ratio with NK cells that are contacted by the Tim-3 inhibitor of the present invention, Examples of protocols for classical cytotoxicity assays are conventional.

As used herein, the term“NK cell exhaustion” refers to a state of NK cell dysfunction. NK cell exhaustion can be defined by poor effector function (i.e. cytotoxic activity), sustained expression of inhibitory receptors, and/or a transcriptional state distinct from that of functional NK cells.

As used herein, the term“asthma” has its general meaning in the art and refers to a chronic disorder characterized by airway inflammation, increased mucus secretion, and bronchial hyperresponsiveness, all of which cause reversible airflow obstruction. The chronic inflammation, disrupted epithelium, and airway remodeling increase the susceptibility to many environmental factors, such as viral infections and allergens. As used herein, the term“severe asthma” has its general meaning in the art and refers to asthma which requires treatment with high doses of corticosteroid and p 2 -adrenergic receptor agonist to prevent it from becoming uncontrolled or which remains uncontrolled despite therapy.

The method of the present invention is particularly suitable for the treatment of exacerbation in a patient suffering from severe asthma.

As used herein, the term“exacerbation” has its general meaning in the art and refers to an acute or subacute episode of progressive worsening of symptoms of asthma, including shortness of breath, wheezing, cough, and chest tightness. Exacerbations are marked by decreases from baseline in objective measures of pulmonary function, such as peak expiratory flow rate and FEV1 (Forced Expiratory Volume). The method of the present invention is thus particularly suitable for reducing the frequency, severity or duration of one or more of said symptoms. In some embodiments, the exacerbation has a viral aetiology. Viral pathogens associated with acute exacerbations in patients with asthma include rhinoviruses (RV), influenza, parainfluenza, coronavirus, adenovirus, and respiratory syncytial virus (RSV). In some embodiments, the exacerbation results from a human rhino virus (HRV) infection. HRVs are nonenveloped positive-strand RNA viruses in the family Picornaviridae and genus Enterovirus and are classified into 3 species (HRV- A, HRV-B and HRV-C). There are more than 160 distinct HRV genotypes, including 80 HRV-A and 32 HRV-B serotypes and 65 newly identified HRV-C serotypes.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term“Tim-3” has its general meaning in the art and refers to T-cell immunoglobulin domain and mucin domain 3 encoded by the HAVCR2 (hepatitis A virus cellular receptor 2) gene (Gene ID: 84868). T cell immunoglobulin-3 (Tim-3) is a direct negative regulator of T cells and is expressed on NK cells and macrophages. The term is also known as TIM-3; CD366; KIM-3; TIMD3; TIMD-3; and HAVcr-2. The amino acid sequence of Tim-3, e.g, human Tim-3, is known in the art, e.g., Sabatos et al, 2003. Nat Immunol,

4(11): 1102 and is represented by SEQ ID NO: 1.

SEQ ID NO : 1 >sp | Q8TDQ0 | HAVR2_HUMAN Hepatitis A virus cellular receptor 2 OS=Homo sapiens OX=9606 GN=HAVCR2 PE=1 SV=3

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPV FECGNWLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMND EKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLA NELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALI FGALIFKWYSHSKEKIQNLSLI SLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAM

P

As used herein, the term“Tim-3 inhibitor” refers to any compound natural or not which is capable of inhibiting the activity of Tim-3. The term encompasses any antagonist that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the Tim-3. The inhibitor can thus inhibit the expression or activity of Tim-3, modulate or block the Tim-3 signalling pathway and/or block the binding of Tim-3 to galectin-9. The term also encompasses inhibitor of expression.

In some embodiments, the Tim-3 inhibitor of the present invention is an antibody, more particularly an antibody having specificity for Tim-3.

As used herein, the term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Rabat et ah, 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161 ; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab’)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab’ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Fe Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody of the present invention is a single chain antibody. As used herein the term“single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also“nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends Biotechnol, 2003, 21(11):484-490; and WO 06/030220, WO 06/003388.

In some embodiments, the antibody is a humanized antibody. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.

In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.

In some embodiments, the antibody of the present invention is a single chain antibody. As used herein the term“single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also “nanobody®”.

In some embodiments, the antibody comprises human heavy chain constant regions sequences but will not induce antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody of the present invention does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD16) polypeptide. In some embodiments, the antibody of the present invention lacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the antibody of the present invention consists of or comprises a Fab, Fab', Fab'-SH, F (ab') 2, Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the antibody of the present invention is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by ldusogie et al.

In some embodiments, the antibody is a multispecific antibody. Multispecific antibodies are typically described in WO2011159877. According to the invention the multispecific antibody of the present invention binds to an extracellular domain of Tim-3. Exemplary formats for the multispecific antibody molecules of the present invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to Tim-3 and another with a specificity to another target of intrestes (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al, Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (viii) a so called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivaient bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (x) a diabody. Another exemplary format for bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), FUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies. In some embodiments, the bispecific antibody is obtained or obtainable via a controlled Fab- arm exchange, typically using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in W02008119353 and WO 2011131746 (both by Genmab A/S). In one exemplary method, described in WO 2008119353, a bispecific antibody is formed by "Fab-arm" or "half- molecule" exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences. In another exemplary method, described in WO 2011131746, bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is a antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is a antibody of the present invention and the second antibody has a different binding specificity, or vice versa. The reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. Step d) may further comprise restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting. Preferably, the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety.

Antibodies having specificity for Tim-3 are well known in the art and typically those described in WO2011155607, W02013006490, WO2010117057 and WO2015117002 and may typically selected from the group consisting of TSR-022, LY3321367 and MBG453. TSR- 022 (Tesaro) is an anti-Tim-3 antibody which is being studied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-Tim-3 antibody which is being studied in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-Tim-3 antibody which is being studied in advanced malignancies (NCT02608268).

In some embodiments, the antibody comprises:

(a) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 2 (GYTFTSY); a VHCDR2 amino acid sequence of SEQ ID NO: 3 (YPGNGD); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 5 (SESVEYYGTSL), a VLCDR2 amino acid sequence of SEQ ID NO: 6 (AAS), and a VLCDR3 amino acid sequence of SEQ ID NO: 7 (SRKDPS);

(b) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 8 (SYNMH); a VHCDR2 amino acid sequence of SEQ ID NO: 9 (DIYPGNGDTSYNQKFKG); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 10 (RASES VEYYGTSLMQ), a VLCDR2 amino acid sequence of SEQ ID NO: 11 (AASNVES), and a VLCDR3 amino acid sequence of SEQ ID NO: 12 (QQSRKDPST);

(c) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 2 (GYTFTSY); a VHCDR2 amino acid sequence of SEQ ID NO: 13 (YPGSGD); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 5 (SESVEYYGTSL), a VLCDR2 amino acid sequence of SEQ ID NO: 6 (AAS), and a VLCDR3 amino acid sequence of SEQ ID NO: 7 (SRKDPS);

(d) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 8 (SYNMH); a VHCDR2 amino acid sequence of SEQ ID NO: 14 (DIYPGSGDTSYNQKFKG); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 10 (RASES VEYYGTSLMQ), a VLCDR2 amino acid sequence of SEQ ID NO: 11 (AASNVES), and a VLCDR3 amino acid sequence of SEQ ID NO: 12 (QQSRKDPST);

(e) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 2 (GYTFTSY); a VHCDR2 amino acid sequence of SEQ ID NO: 15 (YPGQGD); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 5 (SESVEYYGTSL), a VLCDR2 amino acid sequence of SEQ ID NO: 6 (AAS), and a VLCDR3 amino acid sequence of SEQ ID NO: 7 (SRKDPS); or

(f) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO: 8 (SYNMH); a VHCDR2 amino acid sequence of SEQ ID NO: 16 (DIYPGQGDTSYNQKFKG); and a VHCDR3 amino acid sequence of SEQ ID NO: 4 (VGGAFPMDY); and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 10 (RASES VEYYGTSLMQ), a VLCDR2 amino acid sequence of SEQ ID NO: 11 (AASNVES), and a VLCDR3 amino acid sequence of SEQ ID NO: 12 (QQSRKDPST).

In some embodiments, the Tim-3 inhibitor is a polypeptide comprising a functional equivalent of Tim-3 respectively. As used herein, a“functional equivalent of Tim-3” is a polypeptide which is capable of binding to a Tim-3 ligand (e.g. galectin 9), thereby preventing its interaction with Tim-3. The term "functional equivalent" includes fragments, mutants, and muteins of Tim-3. The term "functionally equivalent" thus includes any equivalent of Tim-3 obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to a Tim-3 ligand (e.g. galectin 9). Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence. Functional equivalents include molecules that bind a Tim-3 ligand (e.g. galectin 9) and comprise all or a portion of the extracellular domains of Tim-3 so as to form a soluble receptor that is capable to trap a Tim-3 ligand (e.g. galectin 9). Thus the functional equivalents include soluble forms of the Tim-3. A suitable soluble form of these proteins, or functional equivalents thereof, might comprise, for example, a truncated form of the protein from which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods. Typically, the functional equivalent is at least 80% homologous to the corresponding protein. In a preferred embodiment, the functional equivalent is at least 90% homologous as assessed by any conventional analysis algorithm. The term "a functionally equivalent fragment" as used herein also may mean any fragment or assembly of fragments of Tim-3 that binds to a Tim-3 ligand (e.g. galectin 9). Accordingly the present invention provides a polypeptide capable of inhibiting binding of Tim-3 to a Tim-3 ligand, which polypeptide comprises consecutive amino acids having a sequence which corresponds to the sequence of at least a portion of an extracellular domain of Tim-3, which portion binds to a Tim-3 ligand (e.g. galectin 9). In some embodiments, the polypeptide comprises a functional equivalent of Tim-3 which is fused to an immunoglobulin constant domain (Fc region) to form an immunoadhesin. Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. The immunoglobulin sequence typically, but not necessarily, is an immunoglobulin constant domain. The immunoglobulin moiety in the chimeras of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but typically IgGl or IgG3. In some embodiments, the functional equivalent of the PD-l or Tim-3 and the immunoglobulin sequence portion of the immunoadhesin are linked by a minimal linker. As used herein, the term“linker” refers to a sequence of at least one amino acid that links the polypeptide of the invention and the immunoglobulin sequence portion. Such a linker may be useful to prevent steric hindrances. In some embodiments, the linker has 4; 5; 6; 7; 8; 9; 10; 11 ; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30 amino acid residues. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine linker sequences.

In some embodiments, the Tim-3 inhibitor is an inhibitor of Tim-3 expression respectively. An“inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of Tim-3 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Tim-3, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions ofthe mRNA transcript sequence encoding Tim- 3 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Tim-3 gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Tim-3 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing T im-3. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor of expression is an endonuclease. The term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, and cleave only at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR- cas. As used herein, the term“CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provote lla and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

According to the invention, the Tim-3 inhibitor is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subj ect; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically the active ingredient of the present invention (e.g. Tim-3 inhibitor) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. In some embodiments, the pharmaceutical composition of the invention is administered topically (i.e. in the respiratory tract of the subject). Therefore, the compositions can be formulated in the form of a spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. If the method of the invention comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, the active ingredients for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: NK cells from severe asthma patients exhibit a defective activation following PBMC stimulation with molecules mimicking microbial compounds.

PBMC from severe asthma patients and healthy donors were stimulated with IL-12+IL- 15, FSL1 (TLR2/6 agonist), polyinosinic-polycytidylic acid (PIC, TLR3 agonist), resiquimod (R848, TLR7/8 agonist), CpG oligonucleotides (CpG, TLR9 agonist), or hemagglutinin A of influenza A (HA, recognized by NKp46) for 24 hours. (A) Percentage of CD69 and (B) CD 107a positive NK cells. (C) Concentration of Granzyme B in PBMC supernatants. Horizontal lines represent the median, boxes the interquartile range and whiskers the range. Statistical comparisons between medium condition and stimulated conditions were performed with a Kruskal Wallis test (£r<0.0001, lr<0.01, wr<0.05). Healthy donors (n=8, white) and severe asthma patients (n=l4, black) were compared with a Mann-Whitney test (*p<0.05, ***p<0.00l).

Figure 2: Decreased production of IFN-g following PBMC stimulation with IL- 12+IL-15 but increased production of CXCL9 in severe asthma patients.

PBMC from severe asthma patients and healthy donors were stimulated with IL-12+IL- 15, FSL1 (TLR2/6 agonist), polyinosinic-polycytidylic acid (PIC, TLR3 agonist), resiquimod (R848, TLR7/8 agonist), CpG oligonucleotides (CpG, TLR9 agonist), or hemagglutinin A of influenza A (HA, recognized by NKp46) for 24 hours. (A) Concentrations of IFN-g (pg/ml) in supernatant. (B) Intracellular expression of IFN-g in NK cells expressed as percentage of positive NK cells. (C) Concentration of CXCL9 and (D) CXCL10 (pg/ml). Horizontal lines represent the median, boxes represent the interquartile range and whiskers represent the range. Statistical comparisons between medium condition and stimulated conditions were performed with a Kruskal Wallis test (£r<0.0001, $r<0.001, lr<0.01, wr<0.05). Healthy donors (n=8, white) and severe asthma patients (n=l4, black) were compared with a Mann- Whitney test (*p<0.05).

Figure 3: NK cells from severe asthmatic patients express and produce less IFN-g after stimulation with IL-12+IL-15

(A) PBMC from severe asthmatic patients and healthy donors were stimulated with IL- 12+IL-15 for 24 hours. NK cells positive for IFN-g were identified by flow cytometry. Values are expressed as percent of NK cells among PBMC positive for IFN-g. n=8 healthy donors and n=l4 severe asthmatic patients. (B) Percentage of IFN-y+ NK cells and (C) concentration of IFN-g in NK cell supernatant. Horizontal lines represent the median, boxes represent the interquartile range and whiskers represent the range. Statistical comparisons between medium and ILl2/l5 condition were performed with a Mann- Whitney test (£r<0.0001, lr<0.01). Values are expressed as percent of NK cells positive for IFN-g and as pg/ml. n=8 healthy donors (white) and n=9 (black) severe asthmatics patients.

Figure 4: NK cells from severe asthma patients exhibit decreased cytotoxic status and IFN-g expression following PBMC stimulation with rhinovirus RV-A9 (HRV).

PBMC from healthy donors (n=6, white) and severe asthma patients (n=9, black) were stimulated with RV-A9. (A) Percentage of CD69 and (B) CD 107a positive NK cells. (C) Granzyme B secretion in PBMC supernatants. (D) Percentage of IFN-y+ NK cells and (E) concentration of IFN-g in PBMC supernatant. Horizontal lines represent the median, boxes represent the interquartile range and whiskers represent the range. Statistical comparisons between medium and RV-A9 condition were performed with a Mann- Whitney test (£p<0.000l, lr<0.01, wr<0.05). Healthy donors and severe asthma patients were compared with a Mann- Whitney test (**p<0.0l, *p<0.05).

Figure 5: Increased Tim3 expression restricted to NK cells from severe asthma patients.

PBMC were obtained from healthy donors (n=7) and severe asthma patients (n=l5), and stained with Tim3 specific monoclonal antibody or control isotype antibody. One representative histogram is shown for NK cell staining in (A) for healthy donors and (B) for severe asthma patients, with Tim3 (1) and control isotype (2). Mean Fluorescence Intensity (MFI) values are shown for Tim3 and isotype antibodies for NK cells from healthy donors (C) and severe asthma patients (D). Ratio MFI Tim3 / MFI Isotype was compared for NK cells between healthy subjects (H) and severe asthma patients (SA) (E). In parallel, MFI values are shown for Tim3 and isotype antibodies for T cells from healthy donors (F) and severe asthma patients (G). Ratio MFI Tim3 / MFI Isotype was compared for T cells between healthy subjects and severe asthma patients (H). Horizontal lines represent the median, boxes represent the interquartile range and whiskers represent the range. *p<0.05, ****p<0.0001 (Wilcoxon test for paired isotype versus Tim3, or Mann- Whitney test for difference between healthy and severe asthma subjects).

Figure 6. Neutralization of Tim3 restores rhinovirus-induced NK cell activation.

Peripheral blood mononuclear cells from two severe asthmatic patients were purified using Ficoll Paque-Plus. The expression of Tim3 on NK cells was measured by flow cytometry (percentage of NK cells positive for Tim3 indicated on A: patient 1, and B: patient 2). Mononuclear cells were stimulated during 24h with RVA9 rhinovirus. Four hours before the end of the stimulation, K562 target cells (at different ratios NK cells/target cells) and anti- CD 107a antibody were added to evaluate NK cell cytotoxicity (C-F). One hour before stimulation with RVA9 rhinovirus, and lh before adding target cells, anti-Tim3 neutralizing antibody (clone F38-2E2, circle line) or its isotypic control (square line) were added to the culture (20pg/ml). NK cells positive for CD 107a (percentage: C-D; mean fluorescence intensity E-F) and IFN-g (G-H) were identified by flow cytometry.

EXAMPLE:

Material & Methods

Patient characteristics

Twenty-three severe asthmatic patients were recruited from the Division of Pneumology and Immunoallergology at CHRU of Lille. The project received the approval from the Oohiίΐέ de Protection des Personnes Nord Ouest. All donors signed an informed consent form. Severe asthma was defined according to guidelines from the Severe Asthma Research Program based on ATS/ERS criteria 2 . Data collected at enrolment included the number of exacerbations during the last year, respiratory allergies, the rate of peripheral blood eosinophils, the Forced Expiratory volume in one second (FEV1) and the corticosteroid treatments (Table 1). Severe asthmatic patients were compared to non-atopic non-asthmatic donors (Etablissement Frangais du Sang).

Table 1: Donor characteristics. Age, eosinophil rate and number of exacerbations during the last year are expressed as median, showing minimum and maximum values. The data: Allergy, and Inhaled or Oral corticosteroids are expressed as number among the total number of donors.

PBMC and NK cell isolation

Peripheral blood mononuclear cells (PBMC) were isolated from heparinised venous blood on Ficoll-Paque Plus (Sigma- Aldrich, St Louis, USA). 2.10 6 PBMC/ml were cultured in l .5mL of RPMI containing 2mM L-glutamine, lOOU/ml penicillin, 100 pg/ml streptomycin, 10% Fetal Bovin Serum (Eurobio, Courtaboeuf, France). NK cells were purified from PBMC by immunomagnetic separation (STEMCELL Technologies, Vancouver, Canada). The purity of NK cells was assessed by flow cytometric analysis using antibody against CD3, CD56 (Biolegend, San Diego, USA), CD14 and CD19 (Beckton Dickinson Biosciences, Franklin Lakes, USA). The purity of isolated NK cells was >95%.

Rhinovirus production

Vero cell line (ATCC CCL-81) was grown in Dulbeco's Modified Eagle’s Medium (DMEM, Life Technologies, France) containing 10% fetal bovine serum (FBS, Life Technologies, France). Human rhinovirus 9 (HRV, ATCC VR-489) was propagated in Vero cells in DMEM supplemented with 2% FBS. The infected cells were frozen and thawed three times, then they were centrifuged at 3500 rpm for 10 min, afterwards the supernatant was harvested and used as virus stock stored at -80°C. The viral titer in supernatants of infected cells was assessed using the end-point dilution assay, and the Spearman-Karber statistical method was used to determine the tissue culture 50% infectious dose (TCID50). The infectious titer of virus stock was 1.44 x 10 7 TCID50/ml.

Cell stimulation

PBMC or purified NK cells were stimulated in 24-well plates with Interleukin(IL)-l2 (lOng/ml, Peprotech, Rocky Hill, USA) and IL-15 (lOng/ml, Miltenyi, Bergish Gladbach, Germany), or with an agonist for TLR3: Polyinosinic-polycytidylic acid (PIC) (lOpg/ml, Invivogen, San Diego, USA), for TLR9: ODN 2395 (lOpg/ml, Invivogen, San Diego, USA), for TLR7/8: Resiquimod (R848, lpg/ml, Invivogen, San Diego, USA), for TLR2/6: FSL1 ( 1 Lig/ml, Invivogen, San Diego, USA), for NKp46: Hemagglutinin A of Influenza A (lpg/ml, Interchim, San Diego, USA), or with HRV at Multiplicity Of Infection (MOI) 0.1 during 24 hours at 37°C in humid atmosphere saturated with 5% C0 2 . For the analysis of NK cell degranulation, PBMC were cultured with K562 myeloid tumour cells (ATCC) at a ratio of 100 PBMC for 1 K562 for 3 hours at 37°C in 5% C0 2 . After stimulation, cells were washed with Phosphate Buffered Saline (PBS) and supernatants were collected and stored at -80°C.

Flow cytometry analysis

The following antibodies were used (clone noted in parentheses) : anti-CD3 PECy7 (OKT3), anti-CD56 BV421 (HCD56), anti-CD56 BV510 (HCD56), anti-Tim-3 PE (F38-2E2), anti-PDl PerCP Cy5.5 (NAT105) all from Biolegend (San Diego, USA), anti-CD69 FITC (FN50) and mouse IgGl k FITC Isotype control (MOPC-21), anti-IFN-g PerCP Cy5.5 (B27) and anti-mouse IgGl k PerCP Cy5.5 isotype control (MOPC-21) all from Beckton Dickinson Biosciences (Franklin Fakes, USA), anti-CDl07a APC ef660 (H4A3) and anti-mouse IgGl k APC ef660 isotype control (P3.6.2.8.1), anti-CD62F APC ef780 (DREG56), from Fife Techno logies-Ebio science (Carlsbad, USA), The viability of the cells was assessed using Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego, USA).

After stimulation, PBMC were stained with the viability marker during 20 minutes at room temperature before staining with extracellular antibodies during 30 minutes at 4°C. For intracellular staining, cells were incubated with Cytofix/Cytoperm (Beckton Dickinson Biosciences, Franklin Fakes, USA) during 20 minutes at 4°C before staining with intracellular antibodies during 30 minutes at 4°C. For the analysis of CD 107a expression by NK cells, PBMC were cultured with target cells together with antibody against CD 107a and monensin (Fife Technologies Ebioscience, Carlsbad, USA), and subsequently stained.

Data were acquired on a Canto II flow cytometer (Beckton Dickinson Biosciences, Franklin Fakes, USA) and analyzed with FlowJo software. Natural Killer cells were identified as a lymphoid population that lacked CD3 expression and expressed CD56. Natural killer cell activation was identified based on the expression of CD69, CD 107 and IFN-g.

Cytokine and Chemokine measurement

25 cytokines (GM-CSF, IFN-a, IFN-g, IE-1b, IF-2, IF-2R, IF-4, IF-5, IF-6, IF-7, IF-8, IF- 10, IF-l2(p40/p70), IF- 13, IF- 15, IF- 17, TNF-a, eotaxin, CCF2, CCF3, CCF4, CCF5, CXCF9, CXCF10) were dosed in culture supernatants by Fuminex assay (Fife Technologies) according to the manufacturer’s intructions. After screening cytokines and chemokines in supernatants, EFISA for CXCF9, CXFC10, granzyme B, IF-8 and IFN-g (R&D Systems, Minneapolis, USA) were performed according to the manufacturer’s instructions. Statistical analysis

Statistical analysis was performed using GraphPad Prism software. Mann- Whitney test was used p < 0.05 was considered statistically significant.

Results

Lower activation and cytotoxic status of Natural Killer cells from severe asthmatic patients following PBMC stimulation with pathogen-like molecules

We first evaluated if severe asthmatic status modified NK cell percentage among PBMC. Before stimulation, the mean proportion of peripheral NK cells among PBMC was 11.38% ± 1.82% in healthy donors and 11.06% ± 5.29% in severe asthmatic patients. Proportion ofNK cells were not modified by the stimulation of PBMC with pathogen- like molecules (data not shown). Similarly, no difference was seen in the proportions of the two main subsets ofNK cells: the percent of NK cell CD56 bnght was 7.15% ± 2.65% in healthy subjects and 7.73% ± 3.48% in severe asthmatic donors and was not modified by the stimulation with pathogen- like molecules.

The effect of PBMC stimulation with molecules mimicking microbes on NK cell activation was assessed by the expression of CD69 (Figure 1 A). Following the stimulation with the cytokine cocktail IL-l2±IL-l5, or with PIC, R848 and CpG, the proportion of NK cells positive for CD69 significantly increased compared to the medium condition, suggesting that NK cells were activated. In response to PIC, the percentage ofNK cells CD69 + was significantly lower in severe asthmatic patients compared to healthy donors. The cytotoxicity of NK cells was evaluated with the expression of CD 107a and the release of granzyme B in PBMC supernatants after 3-hours culture with K562 target cells (Figure IB and 1C). After PBMC stimulation with IL-l2±IL-l5, PIC, R848 and CpG, NK cells showed signs of degranulation as the proportion ofNK cells positive for CD 107a significantly increased compared to the medium condition. In supernatants, the concentration of granzyme B was significantly increased in response to IL-l2±IL-l5 and R848, and not significantly in response to PIC (194.8 pg/ml ± 58.75 pg/ml in medium compared to 286 pg/ml ± 49.96 pg/ml with PIC stimulation). In response to IL-l2±IL-l5, PIC and R848, the percentage of CDl07a + NK cells significantly decreased in severe asthmatic patients compared to healthy donors. The concentration of granzyme B was only diminished in the supernatants of PBMC from severe asthmatic patients after PIC stimulation, compared to healthy donors. These results indicate that NK cells from severe asthmatic patients are less activated and exhibit impaired functions in response to PIC, which mimic viral RNA, and in response to cytokine cocktail that stimulates NK cells. Increased CXCL9 and IL-8 production by PBMC, but lower production of IFN-yby NK cells from severe asthmatic patients

Because NK cell from severe asthmatic patients present a defect in cytotoxic response to IL-12+IL-15 and PIC, we focused on the effect of these stimuli on the cytokine and chemokine release in PBMC supernatants. In response to IL-12 and IL-15 and to PIC, IL-1RA, IL-6, IL-15, IP- 10, CCL2, CCL3, CCL4 and TNF-a were detected but their concentrations were not modified between healthy donors and severe asthmatic patients. Th2 cytokines (IL-4, IL-5 and IL-13) and Thl7 (IL-17A and IL-17F) were not detected in PBMC supernatants. IFN-a and IL-12 were produced by PBMC but neither stimulation modified their concentration compared to the medium condition, both in healthy or severe asthmatic donors.

PBMC stimulation with IL-12+IL-15 increased the concentration of interferon- inducible chemokines, CXCL9 and CXCL10 (Figure 2C and 2D), but not of IL-8 (Data not shown). PBMC from severe asthmatic patients significantly produced higher levels of CXCL9 and IL-8 compared to healthy donors, in response to IL-12+IL-15. Purified NK cells from severe asthmatic patients produced IL-8 without any further stimulation than being in culture, suggesting that NK cells may be partly responsible for increased production in severe asthmatic patients. However, IL-12+IL-15 did not enhance this production (Data not shown).

IFN-g is the main cytokine expressed and rapidly produced by NK cells following their activation. In response to IL-12+IL-15, the production of IFN-g significantly increased in PBMC supernatants compared to the medium condition (Figure 2A). The concentration of IFN- g was lower in supernatants from severe asthmatic patients compared to healthy donors. NK cells were one of the cellular sources of IFN-g, as IL-12+IL-15 stimulation significantly increased the percentage of IFN-y+ NK cells both in healthy and severe asthma patients (Figure 2B), although the percentage of IFN-y+ NK cells was significantly lower in severe asthma patients than in controls.

To evaluate if NK cells may participate to this deficient production, the expression of IFN-g was analyzed by flow cytometry in NK cells among PBMC (Figure 3A). Following the stimulation of PBMC with IL-12+IL-15, the proportion of NK cells positive for IFN-g significantly increased in healthy donors and severe asthmatic patients but was significantly lower in severe asthmatic patients compared to healthy subjects. To evaluate if this defect was intrinsic to NK cells, purified NK cells were stimulated with IL-12+IL-15. Neither activation, nor IFN-g production, was induced with PIC alone on purified NK cells (Data not shown). Cytokine stimulation significantly increased the proportion of IFN-y + NK cells (Figure 3B) and IFN-g concentration in supernatants of purified NK cells (Figure 3C), both in healthy donors and severe asthmatic patients. However, IFN-g NK cells and IFN-g concentration was significantly diminished in severe asthmatic patients compared to healthy subjects, thus confirming that NK cells from severe asthmatic patients present an intrinsic defect leading to a decreased expression and production of IFN-g.

Altogether, these results suggest that NK cells from severe asthmatic patients may also be deficient in their ability to produce the anti-viral cytokine IFN-g after non-specific stimulation with cytokine cocktail.

Deficient responses of Natural Killer cells from severe asthmatic patients after in vitro HRV stimulation

Human rhino viruses have been shown to induce immune response through the activation of a combination of innate receptors including TLR3 and TLR7/8 14 . Moreover, asthma exacerbations are due to infection with live HRV. Therefore, to closely mimic the pathophysiological circumstances, PBMC were stimulated with the human rhino virus 9 (HRV). Several concentrations of HRV (MOI 0.01, 0.05, 0.1 and 1) have been tested with 2 timings of stimulation (24h and 48h). The lowest dose (0.1 MOI), and timing (24 hours) which significantly increased CD69 expression on NK cells were chosen for the rest of the study (data not shown). HRV significantly increased the proportion of CD69 + NK cells both in healthy and severe asthmatic donors (Figure 4A), however the percentage of NK cells CD69 + was slightly lower in severe asthmatic patients (67.09% ± 6.02%) compared to healthy donors (81.05% ± 3.54%). The proportion of CDl07 + NK cells significantly increased following the stimulation of PBMC from both type of patients with HRV, indicating that NK cells have degranulated. In parallel, granzyme B release increased in PBMC supernatants (Figure 4C). In severe asthmatic patients, the proportion of CDl07 + NK cells and the concentration of granzyme B was significantly lower compared to healthy donors (Figure 4B, 4C). Similarly, HRV increased IFN-g expression by NK cells and IFN-g production in both type of patients, but significantly less in severe asthmatic patients (Figure 4D, 4E). Altogether, these results suggest that NK cells from severe asthmatic patients are deficient in their activation, cytotoxicity, and IFN-g production in response to HRV.

Natural Killer cells from severe asthmatic patients exhibit exhausted phenotype

To determine the origin of the impaired functions of NK cells in severe asthmatic patients, the phenotype of NK cell was analyzed by flow cytometry before their stimulation. TLR3, TLR7 and TLR9 were not detected on NK cells, and TLR2 was equally detected on NK cells from healthy donors and severe asthmatic patients (data not shown). CD57 is a marker expressed by mature NK cells: its expression was not modified between NK cells from healthy donors and severe asthmatic patients (Data not shown). Increasing number of studies suggest that in chronic infections, NK cells can become functionally exhausted. The checkpoint molecules or exhaustion markers NKG2A and PD-l were detected on NK cells but their expression was not modified between healthy donors and severe asthmatic patients. In contrast, Tim3 expression by NK cells was significantly increased in severe asthma patients compared to healthy donors (Figure 5A-5E). This was restricted to NK cells as Tim3 expression by CD3+T cells was identical in healthy donors and severe asthma patients (Figure 5F-5H).This finding suggests that NK cells from severe asthmatic patients may be exhausted, which may partially account for their impaired functions in response to TLR agonist and HRV stimulation.

Neutralization of Tim3 restores rhinovirus-induced NK cell activation.

Peripheral blood mononuclear cells from two severe asthmatic patients were purified and the expression of Tim3 on NK cells was measured by flow cytometry (Figures 6A-B). Mononuclear cells were then stimulated during 24h with RVA9 rhinovirus. Four hours before the end of the stimulation, K562 target cells and anti-CD 107a antibody were added to evaluate NK cell cytotoxicity and production of IFN-g in presence or not of an anti-Tim3 neutralizing antibody. We show that neutralization of Tim-3 restores rhinovirus-induced NK cell activation especially in patient 1 (Figure 6C-H).

Discussion:

In this study, we explored NK cell functions in severe asthmatic patients and healthy donors in response to stimulation either with molecules mimicking viruses or with a live virus. We chose to stimulate mononuclear cells in order to allow accessory cells to interact with NK cells.

Ex vivo and after 24 hours of culture, we observed that the proportion of NK cells or major subsets like CD56 bnght and CD56 dim was not modified in the peripheral blood between severe asthmatic patients and healthy donors. The percentage of CD56 bnght NK cells was shown to be reduced in peripheral blood leukocytes isolated from patients suffering from allergic rhinitis and intermittent asthma compared to healthy donors 32 . However, the isolation technique in this study analyzed NK cells after a plastic adherent step which may have modified the subset repartition, in contrast to our experimental settings. More recently, NK cells were identified in the bronchoalveolar lavage from severe asthmatic patients and found to be decreased relative to CD4 + T cells. Bronchoalveolar lavage NK cells were skewed toward CD56 dim phenotype in severe asthma 21 , suggesting that NK cells may be differentially regulated or recruited in this specific compartment. In order to mimic HRV stimulation on human PBMC, we first used synthetic agonists for TLR2, TLR3, and TLR7/8, receptors which are involved in HRV-induced immune response 14 . Positive control for NK cell activation was achieved with IL12+IL-15 stimulation, and comparison was made with the stimulation of another known TLR that recognizes viral nucleic acids (TLR9) and of a natural cytotoxic receptor (NKp46). Stimulation with IL-12+IL- 15 or through TLR involved in nucleic acid recognition lead to significant NK cell activation and/or degranulation in both donors, as evidenced by CD69 and CD 107 increased expression, respectively. CD69 was originally described as an early activation marker but may potentially regulate other functions like tissue retention and metabolism of expressing cells 33 . In our experimental settings, high percentage of NK cells expressed CD69. Both observations may account for differences seen in modifications of proportions of NK cells expressing CD69 and CD 107. Interestingly, the evaluation of the cytotoxic function of NK cells through a marker of degranulation indicated that NK cells from severe asthmatic patients had an impaired cytotoxic function after the stimulation with IL-12+IL-15 or TLR involved in HRV immune response (TLR3 and TLR7/8). Our results are consistent with those previously published showing that peripheral blood NK cells from severe asthmatic patients had impaired killing of K562 myeloid target cells after IL-2 stimulation 21 . Although NK cells are not the unique source of granzyme B in PBMC, decreased release of this cytotoxic molecule after TLR3 stimulation further support a defect of NK cells from severe asthmatic patients in their cytotoxic function. Defective cytotoxicity of NK cells from severe asthmatics after stimulation through TLR3 and TLR7/8, both involved in HRV immune response, are related to decreased cytotoxicity (concordance of CD 107 expression and granzyme B release) after activation with HRV9. The defective cytotoxicity of NK cells is consistent with the increased viral load observed in asthmatic patients during infection 34 .

We also measured multiple cytokines and chemokines in the supernatant of PBMC, as an attempt to measure which cytokine/chemokine may be differentially produce or regulated by NK cells from severe asthmatic patients. Most of them were undetected, probably due to short timing of analysis. In both type of donors, the two interferon-induced chemokines, CXCL9 and CXCL10, were increased after IL-12+IL-15 stimulation only. Stimulation of PBMC from severe asthmatics showed elevated CXCL9 production compared to healthy individuals. CXCL9 is produced by a variety of cells and exerts its chemo tactic activity through CXCR3, particularly expressed by NK cells. CXCL9 was shown to be increased in bronchoalveolar lavages from asthmatic patients 35 and increased mRNA expression was found after in vitro stimulation of epithelial cells with HRV 36 . The relationship between potential increase in NK cell recruitment but decrease in their cytotoxic capacity after viral infection in severe asthmatic patients remains to be elucidated. IL-8 levels in IL-12+IL- 15 -activated PBMC were also found significantly increased in severe asthmatics. This is concordant with increased levels of this chemotactic cytokine in bronchoalveolar lavage from asthmatic patients 35 . However, IL-12+IL-15 did not enhance IL-8 production by purified NK cells, suggesting that NK cells may not be involved in the observed changes. In contrast, increased IFN-g release in PBMC supernatants after IL-12+IL-15 stimulation was significantly lower in severe asthmatic patients; and this paralleled decreased intracellular IFN-g expression in NK cells. Similarly, NK cells from patients suffering from atopic dermatitis express less IFN-g following PBMC stimulation with PMA and ionomycin 37 . The role of NK cells in IFN-g decreased production in severe asthmatics is supported by the decreased expression and production by purified NK cells, suggesting that NK cell defect is at least partly intrinsic. This defect was also observed after live HRV stimulation of PBMC from severe asthmatic patients.

We were not able to measure activation, degranulation nor IFN-g production after TLR or HRV stimulation of purified NK cells, suggesting the involvement of other cells (cell-to-cell contact or cytokines), as previously described. Therefore, beside intrinsic defect, the origin of impaired NK cell functions in severe asthmatics may also be extrinsic. The defect of plasmacytoid dendritic cells from asthmatic patients to produce IFN-a 38 is in favor of this hypothesis. However, neither type I IFN nor IL-12, IL-15 or IL-18 were detected in PBMC supernatants after TLR or HRV stimulation. These cytokines may be used by cells or not involved.

Because of our results with IFN-g, we focused on the origin of the intrinsic defect of NK cells from severe asthmatic patients. We first analyzed TLR expression on ex vivo NK cells but could not detect TLR3, TLR7 nor TLR9 by flow cytometry (data not shown). All NK cells expressed TLR2, but there was no difference between healthy donors and severe asthmatic patients (data not shown). The differences may not be visible at the protein level, as suggested by a study showing the reduction TLR7 mRNA expression in alveolar macrophages from severe asthmatic patients 39 . Moreover, although TLR3 expression did not differ in bronchial epithelial cells from asthmatic patients compared to healthy donors, the signaling pathway through TLR3 was shown to be impaired in response to rhino virus infection 40 .

Recent evidences suggest that NK cells can become functionally exhausted during chronic infections. In HIV-infected patients, polyfunctional CD62L + NK cells express high levels of Program Cell Death (PD)-l, NKG2A, and T-cell immunoglobulin and mucin-domain containing (Tim)-3. The expression of these markers on NK cell surface is associated with a decreased cytotoxicity and IFN-g expression in response to TLR3 stimulation 41 . Here we showed that the proportion of CD62L + Tim-3 + NK cells as well as the expression level of Tim- 3 on NK cells is significantly higher in severe asthmatic patients compared to healthy donors. Increased expression of Tim-3 was restricted to NK cells. PD-l was not detected and the expression of NKG2A and KIR2DL2/DL3 was not modified between severe asthmatic patients and healthy donors (data not shown). This result suggests that NK cells from severe asthmatic patients may be exhausted, leading to inefficient activation of NK cells in response to HRV.

The involvement of corticosteroids in this defect needs to be questioned. Indeed, treatments for severe asthma are mainly constituted of high doses of inhaled and/or systemic corticosteroids as anti-inflammatory agents. It was shown that intramuscular injection of triamcinolone modifies BAL NK cell phenotype. In vitro treatment of PBMC with dexamethasone significantly inhibited the capacity of NK cells to lyse K562 target cells, both in healthy donors and severe asthmatic patients 21 . In our study, all severe asthmatic patients were treated with inhaled corticosteroids, and 47.8% of them received systemic corticosteroids. In patients with systemic corticosteroid, the proportion of peripheral blood NK cells was diminished compared to patients without systemic corticosteroid and healthy donors (data not shown). However, the cytotoxicity and IFN-g expression were not decreased between patients with and without systemic corticosteroids. We analyzed Tim-3 expression on NK cells after in vitro treatment of mononuclear cells isolated from 3 different healthy donors with increasing doses of dexamethasone. No dose of dexamethasone increased Tim-3 expression, whereas NK cell viability decreased with the highest dose (lpg/ml) of dexamethasone (data not shown). Therefore, although corticosteroids may certainly modify NK cell- induced immune response in severe asthmatic patients, and therefore have adverse effects in HRV-induced exacerbations, our study highly suggest that a defect in NK cell may be related to the severe asthmatic disease and exist before the treatment.

In conclusion, we found that NK cells from severe asthmatic patients have impaired in vitro functions in response to rhino viruses. This defect may be linked to NK cell exhaustion and may participate to virus-induced asthma exacerbations and the results suggest that neutralization of Tim3 can restores rhino virus-induced NK cell activation.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. 1. Lambrecht, B. N. & Hammad, H. The immunology of asthma. Nat. Immunol. 16, 45-56 (2014).

2. Chung, K. F. et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur. Respir. J. 43, 343-373 (2014).

3. Stone, B. et al. Characterizing patients with asthma who received Global Initiative for Asthma steps 4-5 therapy and managed in a specialty care setting. Allergy Asthma Proc. 39, 27-35 (2018).

4. Chastek, B. et al. Economic Burden of Illness Among Patients with Severe Asthma in a Managed Care Setting. J. Manag. Care Spec. Pharm. 22, 848-861 (2016).

5. Dalal, A. A. et al. Dose-Response Relationship Between Long-Term Systemic Corticosteroid Use and Related Complications in Patients with Severe Asthma. J. Manag. Care Spec. Pharm. 22, 833-847 (2016).

6. McDonald, V. M. & Gibson, P. G. Exacerbations of severe asthma. Clin. Exp. Allergy 42, 670-677 (2012).

7. Sykes, A. & Johnston, S. L. Etiology of asthma exacerbations. J. Allergy Clin. Immunol. 122, 685-688 (2008).

8. Edwards, M. R., Bartlett, N. W., Hussell, T., Openshaw, P. & Johnston, S. L. The microbiology of asthma. Nat. Rev. Microbiol. (2012). doi: l0.l038/nrmicro280l

9. Message, S. D. et al. Rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and Thl/2 cytokine and IL-10 production. Proc. Natl. Acad. Sci. 105, 13562-13567 (2008).

10. Wark, P. A. B. Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhino virus. J. Exp. Med. 201, 937-947 (2005).

11. Contoli, M. et al. Role of deficient type III interferon-l production in asthma exacerbations. Nat. Med. 12, 1023-1026 (2006).

12. Laza-Stanca, V. et al. The Role of IL-15 Deficiency in the Pathogenesis ofVirus- Induced Asthma Exacerbations. PLoS Pathog. 7, el002l 14 (2011).

13. Chairakaki, A.-D. et al. Plasmacytoid dendritic cells drive acute exacerbations of asthma. J. Allergy Clin. Immunol. (2017). doi: l0. l0l6/j.jaci.20l7.08.032

14. Jacobs, S. E., Lamson, D. M., St George, K. & Walsh, T. J. Human rhinoviruses. Clin. Microbiol. Rev. 26, 135-162 (2013).

15. Vivier, E., Tomasello, E., Baratin, M., Walzer, T. & Ugolini, S. Functions of natural killer cells. Nat. Immunol. 9, 503-510 (2008). 16. Long, E. O., Sik Kim, H., Liu, D., Peterson, M. E. & Rajagopalan, S. Controlling Natural Killer Cell Responses: Integration of Signals for Activation and Inhibition. Annu. Rev. Immunol. 31, 227-258 (2013).

17. Mandelboim, O. et al. Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature 409, 1055-1060 (2001).

18. Adib-Conquy, M., Scott- Algara, D., Cavaillon, J.-M. & Souza-Fonseca- Guimaraes, F. TLR-mediated activation of NK cells and their role in bacterial/viral immune responses in mammals. Immunol. Cell Biol. 92, 256-262 (2014).

19. Caligiuri, M. A. Human natural killer cells. Blood 112, 461-469 (2008).

20. Bamig, C. et al. Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma. Sci. Transl. Med. 5, l74ra26 (2013).

21. Duvall, M. G. et al. Natural killer cell-mediated inflammation resolution is disabled in severe asthma. Sci. Immunol. 2, eaam5446 (2017).

22. Lin, S.-J. et al. Decreased intercellular adhesion molecule-l (CD54) and L- selectin (CD62L) expression on peripheral blood natural killer cells in asthmatic children with acute exacerbation. Allergy 58, 67-71 (2003).

23. Awad, A. et al. Natural Killer Cells Induce Eosinophil Activation and Apoptosis. PLoS ONE 9, e94492 (2014).

24. Ghadially, H. et al. NKp46 regulates allergic responses. Eur. J. Immunol. 43, 3006-3016 (2013).

25. Korsgren, M. et al. Natural Killer Cells Determine Development of Allergen- induced Eosinophilic Airway Inflammation in Mice. J. Exp. Med. 189, 553-562 (1999).

26. Haspeslagh, E. et al. Role of NKp46 + natural killer cells in house dust mite - driven asthma. EMBO Mol. Med. e8657 (2018). doi: l0.l5252/emmm.201708657

27. Haworth, O., Cemadas, M. & Levy, B. D. NK Cells Are Effectors for Resolvin El in the Timely Resolution of Allergic Airway Inflammation. J. Immunol. 186, 6129-6135 (2011).

28. Simons, B. et al. PGI 2 Controls Pulmonary NK Cells That Prevent Airway Sensitization to House Dust Mite Allergen. J. Immunol. 198, 461-471 (2017).

29. Ferrini, M. E. et al. CB2 receptors regulate natural killer cells that limit allergic airway inflammation in a murine model of asthma. Allergy 72, 937-947 (2017).

30. Lunding, L. P. et al. Poly(inosinic-cytidylic) Acid-Triggered Exacerbation of Experimental Asthma Depends on IL-17A Produced by NK Cells. J. Immunol. 194, 5615-5625 (2015). 31. Cooper, G., Khakoo, S., Wilkinson, T. & Staples, K. LSC - 2017 - Tissue- resident Natural Killer (NK) cell Phenotype in the Human Lung in PA2013 (European Respiratory Society, 2017). doklO.l 183/1393003. congress-2017.PA2013

32. Scordamaglia, F. et al. Perturbations of natural killer cell regulatory functions in respiratory allergic diseases. J. Allergy Clin. Immunol. 121, 479-485 (2008).

33. Cibrian, D., Sanchez-Madrid, F. D69: from activation marker to metabolic gatekeeper. Eur J Immunol. 47(6):946-953 (2017).

34. Kennedy, J. F. et al. Comparison of Viral Foad in Individuals with and without Asthma during Infections with Rhino virus. Am. J. Respir. Crit. Care Med. 189, 532-539 (2014).

35. Hosoki, K., et al. Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IF-8 Fevels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma, and Correlates Inversely with FEV1. PFoS One. l0(5):e0l26035. (2015).

36. Tan, K.S. In Vitro Model of Fully Differentiated Human Nasal Epithelial Cells Infected With Rhinovirus Reveals Epithelium-Initiated Immune Responses. J Infect Dis. 217(6):906-915 (2018).

37. Fuci, C. et al. Peripheral natural killer cells exhibit qualitative and quantitative changes in patients with psoriasis and atopic dermatitis. Br. J. Dermatol. 166, 789-796 (2012).

38. Gill, M. A. et al. Counterregulation between the Fc RI Pathway and Antiviral Responses in Human Plasmacytoid Dendritic Cells. J. Immunol. 184, 5999-6006 (2010).

39. Rupani, H. et al. Toll-like Receptor 7 Is Reduced in Severe Asthma and Finked to an Altered MicroRNA Profile. Am. J. Respir. Crit. Care Med. 194, 26-37 (2016).

40. Parsons, K. S., Hsu, A. C. & Wark, P. a. B. TFR3 and MDA5 signalling, although not expression, is impaired in asthmatic epithelial cells in response to rhinovirus infection. Clin. Exp. Allergy J. Br. Soc. Allergy Clin. Immunol. 44, 91-101 (2014).

41. Fima, J. F., Oliveira, F. M. S., Pereira, N. Z., Duarte, A. J. S. & Sato, M. N. Polyfunctional natural killer cells with a low activation profile in response to Toll-like receptor 3 activation in HIV-l-exposed seronegative subjects. Sci. Rep. 7, 524 (2017).