METHODS FOR THE DIAGNOSIS AND TREATMENT OF CYTOKINE RELEASE SYNDROME

Abstract

The invention relates to methods and pharmaceutical compositions for the treatment of Cytokine Release Syndrome (CRS). The invention also relates to methods for the diagnosis of patients suffering from Cytokine Release Syndrome. The inventors investigate iron homeostasis and the role of CD44-mediated iron endocytosis in the severe inflammatory response and Cytokine Release Syndrome, particularly in SARS-CoV-2 patients. The inventors demonstrate that during activation of M1 macrophages, iron endocytosis is upregulated in a CD44-dependent manner and that CD44 protein levels increase. This effect is specific to M1 macrophages, whereas levels of the canonical iron endocytosis protein TfR1/CD71 remain unchanged. In the present invention, the inventors provide in vitro evidences towards a direct role of CD44-mediated iron endocytosis in the severe inflammatory response such as Cytokine Release Syndrome observed in SARS-CoV-2 patients. Thus, the present invention relates to an antagonist of CD44/Hyaluronic Acid pathway for use in the treatment of cytokine release syndrome in a subject in need thereof, particularly severe COVID-19-related cytokine release syndrome.

Claims

1-8. (canceled)

9. A method for the treatment of cytokine release syndrome in a subject in need thereof comprising administrating a therapeutic effective amount of an antagonist of CD44/Hyaluronic Acid (HA) pathway selected from the group consisting of CD44 antagonists and CD44 expression inhibitors.

10. The method according to claim 9, wherein the cytokine release syndrome is severe COVID-19-related cytokine release syndrome.

11. The method according to claim 9, wherein said CD44 antagonist is selected from the group consisting of a small organic molecule, polypeptide, aptamer and antibody.

12. The method according to claim 11, wherein said antibody is selected from the group consisting of RG7356 and Bivatuzumab.

13. The method according to claim 9, wherein said CD44 expression inhibitor is selected from the group consisting of an antisense oligonucleotide, shRNA, siRNA, RNAi and a ribozyme.

14. The method according to claim 9, wherein the method is a method for the treatment of Respiratory Distress Syndrome (ARDS) in a subject in need thereof.

15. The method according to claim 9, wherein the method is a method for the treatment of Macrophage Activation Syndrome (MAS) in a subject in need thereof.

16. The method according to claim 9, wherein the method is a method for the treatment of iron related inflammatory diseases and alveolar inflammatory responses in a subject in need thereof.

17. The method according to claim 10, wherein said CD44 antagonist is selected from the group consisting of a small organic molecule, polypeptide, aptamer and antibody.

18. The method according to claim 17, wherein said antibody is selected from the group consisting of RG7356 and Bivatuzumab.

19. The method according to claim 10, wherein said CD44 expression inhibitor is selected from the group consisting of an antisense oligonucleotide, shRNA, siRNA, RNAi and a ribozyme.

Description

FIGURES

[0144] FIG. 1: Increase of CD44 and RhoNOX-M in activated macrophages. GM-CSF-treated monocytes (M1) were activated with LPS (100 ng/mL) during 24 hours and CSF1-treated monocytes were activated with IL4 (20 ng/mL) during 24 hours. CD44, TfR and RhoNOX-M were measured by flow cytometry.

[0145] FIG. 2: CD44-dependent iron endocytosis during macrophagic activation

[0146] FIG. 3: CD44 antagonists induce decreased levels of cellular iron in activated monocyte-derived macrophages. (A) ICP-MS of cellular iron and copper in activated (act.) monocyte-derived macrophages (MDM) treated with siCtrl. or siCD44. n=7 donors. (B) Representative western blot of CD44 in act. MDM transfected with siRNA against CD44 or control siRNA. n=4 donors. Mann-Whitney test. Mean values ±SEM. (C) ICP-MS of cellular iron and copper in act. MDM treated with the anti-CD44 antibody RG7356. n=7 donors. Different donors are denoted in each panel with distinctly dots.

EXAMPLE

Example 1

[0147] Material & Methods

[0148] Chemical reagents and antibodies. GM-CSF and IFN-gamma were obtained from Miltenyi (Miltenyi Biotec, Somerville, MA, USA), Deferoxamine (DFO) from Sigma-Aldrich (SIGMA-ALDRICH, Saint-Quentin Fallavier, France), RhoNox-M (in-house), Hyaluronate Fluorescein (FITC-Hyal) (#YH4532, Carbosynth, San Diego, Calif., USA), Transferrin from human serum, Alexa Fluor 647 conjugate (TF-647) (#T23366) (ThermoFisher Scientific, Waltham, Mass., USA). We used antibodies to CD44 (#ab189524, WB: 1:30000, Abcam, Cambridge, Mass., USA), Ferritin (#ab75973, WB: 1:1000, Abcam), H3K9me2 (#4658S, WB: 1:1000, Cell Signaling, Danvers, Mass., USA), Transferrin receptor 1 (TfR1) (#13-6800, WB: 1:1000, Thermo Fisher Scientific).

[0149] Cell culture. Peripheral blood samples were collected from healthy donors. Pan monocytes were sorted using microbeads according to the manufacturer's instructions (Miltenyi Biotec, Somerville, MA, USA), cultured in RPMI 1640 with glutamine (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and exposed to 100 ng/mL GM-CSF to generate their differentiation. After 5 days of differentiation, macrophages were activated with IFN-g (50 ng/mL) and treated by DF0 (10 m) during 24 hours.

[0150] Bronchoalveolar lavages (BAL). BAL from COVID patients were centrifuged, then alveolar macrophages were purified by adherence in plastic dishes in serum-free RPMI 1640 culture medium without serum or antibiotics at 37° C. in an humidified atmosphere containing 5% C02. After approximatively 15-30 min of incubation the nonadherent cells were removed and the layer of adherent cells were washed twice with 10 mL of ice-cold sterile PBS. The adherent cells were treated overnight by DFO or RG7356.

[0151] Cell morphology. Phase contrast images were captured with a CKX41 microscope (Olympus) and cellSens Entry imaging software (Olympus) before manually tracing the long and short axes of each cell (long axis, longest length of the cell; short axis, length across the nucleus in a direction perpendicular to the long axis) to measure the elongation factor as the ratio of these axes.

[0152] Flow cytometry analysis of cell phenotype. Cells were washed with ice-cold PBS, incubated with Fc block (Human TruStain FcX, Biolegend, London, United Kingdom, 1/20) for 15 min, incubated with antibodies for 20 minutes at 4° C. and washed before analysis using a BD LSRFortessa X-20. Antibodies were AlexaFluor700-CD80 (#561133, BD, Franklin Lakes, N.J., USA), PE/Cy7-CD86 (#561128, BD), APC/Alexa750-CD71 (#A89313, Beckman coulter, Brea, Calif., USA), Krome orange-CD14 (#B01175, Beckman coulter), Pacific Blue-CD16 (#A82792, Beckman coulter), PE-CD163 (#556018, BD) and AlexaFluor647-CD44 (#NB500-481AF647, Novus Biologicals). The data were analyzed with FlowJo software v. 10.0.00003.

[0153] Immunofluorescence microscopy. Cells were spotted on slides using a cytospin centrifuge, washed in PBS, fixed with 4% formaldehyde in PBS for 20 minutes, washed with PBS, post-fixed and permeabilized with cold 70% ethanol for 20 minutes, washed with PBS, blocked with 8% bovine serum albumin (BSA) in PBS for 1 hour, incubated with the first antibody in 1% BSA in PBS for 2 hours, washed and incubated with a secondary antibody conjugated with Alexa Fluor 488 or 555 for 1 hour, washed and mounted by using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, Calif., USA).

[0154] Other fluorescence imaging experiments. Live cells were incubated for 1 hour at 37° C. with 5% C02 in medium containing RhoNox-M, FITC-Hyal, TF-647. Then, cells were washed three times with PBS, spotted on slides using a cytospin centrifuge and mounted by using Vectashield mounting medium with DAPI (Vector Laboratories).

[0155] Fluorescence images were acquired using a Deltavision real-time microscope (Applied Precision). 40×/1.4NA, 60×/1.4NA and 100×/1.4NA objectives were used for 2D and 3D acquisitions. Data were deconvoluted with SoftWorx (Ratio conservative—15 iterations, Applied Precision) and processed with ImageJ. All images were acquired as z-stacks.

[0156] Inductively coupled plasma mass spectrometry. Glass vials equipped with Teflon septa were cleaned with nitric acid 65% (VWR), washed with ultrapure water (Sigma-Aldrich) and dried. Cells were plated 24 h prior to the experiments. In all experiments, cells were incubated for 2 h with FBS-free medium prior to treatment. Cells were harvested, followed by two washes with PBS. Cells were then counted using an automated cell counter (Entek) and transferred in 100 μL PBS to clean vials, and samples were lyophilized using a freeze dryer (CHRIST). Samples were subsequently mixed with nitric acid 65% overnight followed by heating at 80° C. for 2 h. Samples were diluted with ultrapure water (Sigma-Aldrich) to a final concentration of 0.475 N nitric acid and transferred to metal-free centrifuge vials (VWR) for subsequent ICP-MS analysis. .sup.56Fe concentrations were measured using an Agilent 7900 ICP-QMS in low-resolution mode. Sample introduction was achieved with a micro-nebulizer (MicroMist, 0.2 mL/min) through a Scott spray chamber. Isotopes were measured using a collision-reaction interface with helium gas (5 mL/min) to remove polyatomic interferences. Scandium and indium internal standards were injected after inline mixing with the samples to control the absence of signal drift and matrix effects. A mix of certified standards was measured at concentrations spanning those of the samples to convert count measurements to concentrations in the solution. Uncertainties on sample concentrations were calculated using algebraic propagation of ICP-MS blank and sample counts uncertainties. Values were normalized by dry weight and cell number.

[0157] Immunoblot analyses. Cells were washed twice in PBS and lysed in 2×Laemmli buffer containing benzonase (#VWR, Fontenay-Sous-Bois, France), extracts were incubated at 37° C. for 1 h, and quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Protein lysates were resolved on Nu-PAGE 4-12% Bis-Tris gels (Invitrogen, Carlsbad, Calif., USA) and electroblotted onto nitrocellulose membranes (Bio-Rad, Hercules, Calif., USA). Membranes saturated with milk were incubated overnight at 4° C. with primary antibodies, then for 45 min with peroxydase-conjugated anti-IgG secondary antibodies (Jackson Laboratories, Bar Harbor, Me., USA). Signals were revealed by autoradiography using the SuperSignal West Pico PLUS or West Femto enhanced chemiluminescent detection kits (Thermo Fisher Scientific).

[0158] Cytokine profile in human macrophage supernatants. IL1 beta, IL4, IL6, IL8, IL10, IL12, IFNgamma and TNF-alpha concentrations were measured in cell culture supernatants using the human Pro-Inflammatory Combo 1 U-Plex (MSD, Rockville, Md., US). The kit was run according to the manufacturer's guidelines and the chemiluminescence signal was measured on a Sector Imager 2400 (MSD).

[0159] RNA sequencing. RNA extracted using the RNeasy mini kit (QIAGEN, Germantown, MD, USA) was processed using SureSelect Automated Strand Specific RNA Library Preparation Kit and sequenced on Illumina Novaseq 6000 in paired-end 100 bp mode in order to reach at least 40 million reads per sample. Gene expression validation will be done by qRT-PCR are listed below.

[0160] Results

[0161] Our unpublished data indicate that during activation of M1 macrophages, iron endocytosis is upregulated in a CD44-dependent manner and that CD44 protein levels increase. Moreover, lysosomal Fe.sup.2+ is increased in activated M1 macrophages as observed by RhoNox-1. This effect is specific to M1 macrophages and levels of the canonical iron endocytosis protein TfR1/CD71 remain unchanged (FIGS. 1 and 2).

Example 2

[0162] Material & Methods

[0163] Cell culture. Peripheral blood samples were collected from distinct healthy donors (Etablissement Frangais du Sang). Pan monocytes were isolated by negative magnetic sorting using microbeads according to the manufacturer's instructions (Miltenyi Biotec, 130-096-537), and cultured in RPMI 1640 supplemented with glutamine (Thermo Fisher Scientific, 61870010), 10% fetal bovine serum and treated with granulocyte-macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec, 130-093-866, 100 ng/mL) to induce differentiation into macrophages (MDM). At day 5 of differentiation, MDM were treated with lipopolysaccharides (LPS, InvivoGen, tlr1-3pelps, 100 ng/mL, 24 h) and interferon-γ (IFNγ, Miltenyi Biotec, 130-096-484, 20 ng/mL, 24 h) to generate activated MDM (act. MDM) and co-treated with anti-CD44 antibody RG7356 or transfected with a control or CD44 siRNA using the AMAXA Nucleofector transfection system (LONZA). Suitable small interfering RNAs for specific down-regulation of CD44 were the followings

TABLE-US-00001 (SEQ ID NO: 1) 5′-GAAUAUAACCUGCCGCUUU-3′, (SEQ ID NO: 2) 5′-CAAGUGGACUCAACGGAGA-3′, (SEQ ID NO: 3) 5′-CGAAGAAGGUGUGUGGGCAGA-3′, and (SEQ ID NO: 4) 5′-GAUCAACAGUGGCAAUGGA-3′.

[0164] Western Blotting. Cells were treated as indicated and then washed with 1×PBS. Proteins were solubilized in 2×Laemmli buffer containing benzonase (VWR, 70664-3, 1:100), extracts were incubated at 37° C. for 1 h, and quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Protein lysates were resolved by SDS-PAGE electrophoresis (Invitrogen sure-lock system and Nu-PAGE 4-12% Bis-Tris precast gels) and transferred onto nitrocellulose membranes (Amersham Protran 0.45 m) using a Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-rad). Membranes were blocked with 5% non-fat skimmed milk powder in 0.1% Tween-20/1× PBS for 1 h. Blots were then probed with the relevant primary antibodies in 5% BSA, 0.1% Tween-20/1× PBS at 4° C. overnight with gentle motion. Membranes were washed with 0.1% Tween-20/1× PBS three times and incubated with horseradish peroxidase conjugated secondary antibodies (Jackson Laboratories) in 5% non-fat skimmed milk powder, 0.1% Tween-20/1× PBS for 1 h at room temperature and washed three times with 0.1% Tween-20/1× PBS. Antigens were detected using the SuperSignal West Pico PLUS chemiluminescent detection kits (ThermoFisher Scientific, 34580 and 34096). Signals were recorded using a Fusion Solo S Imaging System (Vilber) and quantified with ImageJusing pixel intensity normalized against the signal of γ-tubulin.

[0165] Inductively coupled plasma mass spectrometry (ICP-MS). HA (Carbosynth, FH45321, 600-1000 kDa, 1 mg/mL) was added together with LPS and IFNγ and cells were treated for 24 h. Glass vials equipped with Teflon septa were cleaned with nitric acid 65% (VWR, Suprapur, 1.00441.0250), washed with ultrapure water (Sigma-Aldrich, 1012620500) and dried. Cells were harvested followed by two washes with 1× PBS. Cells were then counted using an automated cell counter (Entek) and transferred in 200 μL 1× PBS to the cleaned glass vials, and samples were lyophilized using a freeze dryer (CHRIST, 22080). Samples were subsequently mixed with nitric acid 65% overnight and heated at 80° C. for 2 h. Samples were diluted with ultrapure water to a final concentration of 0.475 N nitric acid and transferred to metal-free centrifuge vials (VWR, 89049-172) for subsequent ICP-MS analysis. Amounts of .sup.56Fe and .sup.63Cu were measured using an Agilent 7900 ICP-QMS in low-resolution mode. Sample introduction was achieved with a micro-nebulizer (MicroMist, 0.2 mL/min) through a Scott spray chamber. Isotopes were measured using a collision-reaction interface with helium gas (5 mL/min) to remove polyatomic interferences. Scandium and indium internal standards were injected after inline mixing with the samples to control the absence of signal drift and matrix effects. A mix of certified standards was measured at concentrations spanning those of the samples to convert count measurements to concentrations in the solution. Uncertainties on sample concentrations were calculated using algebraic propagation of ICP-MS blank and sample counts uncertainties. Values were normalized against cell number.

[0166] Results

[0167] Down-regulation of CD44 using a small interfering RNA (siRNA) against CD44 reduced the total amount of iron in activated monocyte-derived M1 macrophages (act. MDM) compared to the control RNA (FIG. 3A, B). In addition, treatment with a blocking antibody that binds CD44 (RG7356), reduces the amount of total iron in act MDM (FIG. 3C). These data confirmed that CD44 mediates iron uptake during activation of inflammatory macrophages.

[0168] Altogether, these data are hinting towards a direct role of CD44-mediated iron endocytosis in the severe inflammatory response observed in SARS-CoV-2 patients. Institut Curie has considerable experience with the use of RG7356, a clinically approved anti-CD44 antibody, which has shown promising results in cancer patients with poor outcomes (First-in-human phase I clinical trial of RG7356, an anti-CD44 humanized antibody, in patients with advanced, CD44-expressing solid tumors. Oncotarget (2016), DOI: 10.18632/oncotarget.11098). Here, the inventors evaluate the effect of the use of antagonist of CD44/Hyaluronic Acid (HA) pathway and targeting of iron homeostasis such as by using DFO to antagonize IL6 signalling and to protect SARS-CoV-2 patients from CRS.

REFERENCES

[0169] 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.

[0170] Crayne C B, Albeituni S, Nichols K E, Cron R Q. The Immunology of Macrophage Activation Syndrome. Front Immunol. 2019 Feb 1; 10:119. doi:10.3389/fimmu.2019.00119.

[0171] D'Arena G, Calapai G, Deaglio S. Anti-CD44 mAb for the treatment of B-cell chronic lymphocytic leukemia and other hematological malignancies: evaluation of WO2013063498. Expert Opin Ther Pat. 2014 July;24(7):821-8. doi: 10.1517/13543776.2014.915942.

[0172] Davila M L, Riviere I, Wang X, Bartido S, Park J, Curran K, Chung S S, Stefanski J, Borquez-Ojeda O, Olszewska M, Qu J, Wasielewska T, He Q, Fink M, Shinglot H, Youssif M, Satter M, Wang Y, Hosey J, Quintanilla H, Halton E, Bernal Y, Bouhassira D C, Arcila M E, Gonen M, Roboz G J, Maslak P, Douer D, Frattini M G, Giralt S, Sadelain M, Brentjens R. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. 2014 Feb 19;6(224):224ra25. doi: 10.1 126/scitranslmed.

[0173] Hernández-Beeftink T, Guillen-Guio B, Villar J, Flores C. Genomics and the Acute Respiratory Distress Syndrome: Current and Future Directions. Int J Mol Sci. 2019 Aug 16;20(16). pii: E4004. doi: 10.3390/ijms20164004.

[0174] Khan A, Singh P, Srivastava A. Synthesis, nature and utility of universal iron chelator-Siderophore: A review. Microbiol Res. 2018 Jul-Aug;212-213:103-111. doi: 10.1016/j.micres.2017.10.012.

[0175] Kontoghiorghes G J. Comparative efficacy and toxicity of desferrioxamine, deferiprone and other iron and aluminium chelating drugs. Toxicol Lett. 1995 October;80(1-3):1-18.

[0176] Lerkvaleekul B, Vilaiyuk S. Macrophage activation syndrome: early diagnosis is key. Open Access Rheumatol. 2018 Aug 31; 10:117-128. doi: 10.2147/OARRR.S151013.

[0177] Lima T G, Benevides F L N, Esmeraldo Filho F L, Farias I S, Dourado D X C, Fontenele E G P, Moraes M E A, Quidute A R P. Treatment of iron overload syndrome: a general review. Rev Assoc Med Bras (1992). 2019 Oct 10;65(9):1216-1222. doi:10.1590/1806-9282.65.9.1216.

[0178] Maude S L, Barrett D, Teachey D T, Grupp S A. Managing cytokine release syndrome associated with novel T cell-engaging therapies. Cancer J. 2014 Mar-Apr;20(2):119-22. doi: 10.1097/PPO.0000000000000035.

[0179] McKee C M, Penno M B, Cowman M, Burdick M D, Strieter R M, Bao C, Noble P W. Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J Clin Invest. 1996 Nov 15;98(10):2403-13. DOI: 10.1172/JCI119054.

[0180] Menke-van der Houven van Oordt C W, Gomez-Roca C, van Herpen C, Coveler A L, Mahalingam D, Verheul H M, van der Graaf W T, Christen R, Ruttinger D, Weigand S, Cannarile M A, Heil F, Brewster M, Walz A C, Nayak T K, Guarin E, Meresse V, Le Tourneau C. First-in-human phase I clinical trial of RG7356, an anti-CD44 humanized antibody, in patients with advanced, CD44-expressing solid tumors. Oncotarget. 2016 Nov 29;7(48):80046-80058. doi: 10.18632/oncotarget.11098.

[0181] Merad M, Martin J C. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat Rev Immunol. 2020 May 6. doi:10.1038/s41577-020-0331-4.

[0182] Mobarra N, Shanaki M, Ehteram H, Nasiri H, Sahmani M, Saeidi M, Goudarzi M, Pourkarim H, Azad M. A Review on Iron Chelators in Treatment of Iron Overload Syndromes. Int J Hematol Oncol Stem Cell Res. 2016 Oct 1;10(4):239-247.

[0183] Moore J B, June C H. Cytokine release syndrome in severe COVID-19. Science. 2020 May 1;368(6490):473-474. doi: 10.1126/science.abb8925.

[0184] Müller Sebastian, Sindikubwabo Fabien, Caneque Tatiana, Lafon Anne, Versini Antoine, Lombard B6rangere, Loew Damarys, Durand Adeline, Vallot Celine, Baulande Sylvain, Servant Nicolas, Rodriguez Raphael. CD44 regulates epigenetic plasticity by mediating iron endocytosis. bioRxiv 693424; doi: https://doi.org/10.1101/693424. Accepted in Nature Chemistry.

[0185] Niesler U, Palmer A, Radermacher P, Huber-Lang M S. Role of alveolar macrophages in the inflammatory response after trauma. Shock. 2014 July;42(1):3-10. doi: 10.1097/SHK.0000000000000167.

[0186] Ponta, H., Sherman, L. & Herrlich, P. A. CD44: from adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 4, 33-45, (2003).

[0187] Ranieri V M, Rubenfeld G D, Thompson B T, Ferguson N D, Caldwell E, Fan E, Camporota L, Slutsky A S. ARDS Definition Task Force. Acute respiratory distress syndrome: the Berlin Definition. JAMA. 2012 Jun 20;307(23):2526-33. doi:10.1001/jama.2012.5669.

[0188] Shalabi H, Hahn K, Fry T J, Shah N N. Novel Designs of Early Phase Trials for Cancer Therapeutics. Chapter 12—Cell-Based Therapies: A New Frontier of Personalized Medicine. Editor(s): Shivaani Kummar, Chris Takimoto. Academic Press, 2018, Pages 175-191, ISBN 9780128125120, https://doi.org/10.1016/B978-O-12-812512-0.00012-9.

[0189] Shimabukuro-Vornhagen A, Godel P, Subklewe M, Stemmler H J, Schlößer H A, Schlaak M, Kochanek M, Boll B, von Bergwelt-Baildon M S. Cytokine release syndrome. J Immunother Cancer. 2018 Jun 15;6(1):56. doi:10.1186/s40425-018-0343-9.

[0190] Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S, Stein T I, Nudel R, Lieder I, Mazor Y, Kaplan S, Dahary D, Warshawsky D, Guan-Golan Y, Kohn A, Rappaport N, Safran M, Lancet D. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics. 2016 Jun 20; 54:1.30.1-1.30.33. doi: 10.1002/cpbi.5.

[0191] UniProt Consortium. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019 Jan 8;47(D1):D506-D515. doi: 10.1093/nar/gky1049.

[0192] Weigand S, Herting F, Maisel D, Nopora A, Voss E, Schaab C, Klammer M, Tebbe A. Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist. Cancer Res. 2012 Sep 1;72(17):4329-39. doi: 10.1158/0008-5472.CAN-12-0136.

[0193] Whyte C S, Morrow G B, Mitchell J L, Chowdary P, Mutch N J. Fibrinolytic abnormalities in acute respiratory distress syndrome (ARDS) and versatility of thrombolytic drugs to treat COVID-19. J Thromb Haemost. 2020 Apr 23. doi:10.1111/jth.14872.

[0194] Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu X, Guan L, Wei Y, Li H, Wu X, Xu J, Tu S, Zhang Y, Chen H, Cao B. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020 Mar 28;395(10229):1054-1062. doi:10.1016/S0140-6736(20)30566-3. Erratum in: Lancet. 2020 Mar 28;395(10229):1038. Lancet. 2020 Mar 28;395(10229):1038.

[0195] Zhou M, Zhang X, Qu J. Coronavirus disease 2019 (COVID-19): a clinical update. Front Med. 2020 April; 14(2):126-135. doi: 10.1007/s11684-020-0767-8.

[0196] Zöller, M. CD44: can a cancer-initiating cell profit from an abundantly expressed molecule? Nat. Rev. Cancer 11, 254-267, (2011).