A novel platform for attenuating immune hyperactivity using EXO‐CD24 in COVID‐19 and beyond
2022; Springer Nature; Volume: 14; Issue: 9 Linguagem: Inglês
10.15252/emmm.202215997
ISSN1757-4684
AutoresShiran Shapira, Marina Ben Shimon, Mori Hay‐Levi, Gil Shenberg, Guy Choshen, Lian Bannon, Michael Tepper, Dina Kazanov, Jonathan Seni, Shahar Lev‐Ari, Michael Peer, Dimitrios Boubas, Justin Stebbing, Sotirios Tsiodras, Nadir Arber,
Tópico(s)SARS-CoV-2 and COVID-19 Research
ResumoArticle13 July 2022Open Access Transparent process A novel platform for attenuating immune hyperactivity using EXO-CD24 in COVID-19 and beyond Shiran Shapira Shiran Shapira The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Marina Ben Shimon Marina Ben Shimon The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Mori Hay-Levi Mori Hay-Levi The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Gil Shenberg Gil Shenberg The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Guy Choshen Guy Choshen Department of Internal Medicine H, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Search for more papers by this author Lian Bannon Lian Bannon orcid.org/0000-0002-5317-3613 Department of Internal Medicine F, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Search for more papers by this author Michael Tepper Michael Tepper The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Dina Kazanov Dina Kazanov The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Jonathan Seni Jonathan Seni The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Shahar Lev-Ari Shahar Lev-Ari orcid.org/0000-0003-0187-9427 The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Michael Peer Michael Peer Department of Thoracic Surgery, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Search for more papers by this author Dimitrios Boubas Dimitrios Boubas 4th Department of Internal Medicine, Attikon University Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece Search for more papers by this author Justin Stebbing Justin Stebbing orcid.org/0000-0002-1117-6947 Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Sotirios Tsiodras Sotirios Tsiodras 4th Department of Internal Medicine, Attikon University Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece Search for more papers by this author Nadir Arber Corresponding Author Nadir Arber [email protected] orcid.org/0000-0001-5283-6991 The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Shiran Shapira Shiran Shapira The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Marina Ben Shimon Marina Ben Shimon The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Mori Hay-Levi Mori Hay-Levi The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Gil Shenberg Gil Shenberg The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Guy Choshen Guy Choshen Department of Internal Medicine H, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Search for more papers by this author Lian Bannon Lian Bannon orcid.org/0000-0002-5317-3613 Department of Internal Medicine F, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Search for more papers by this author Michael Tepper Michael Tepper The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Dina Kazanov Dina Kazanov The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Search for more papers by this author Jonathan Seni Jonathan Seni The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Shahar Lev-Ari Shahar Lev-Ari orcid.org/0000-0003-0187-9427 The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Michael Peer Michael Peer Department of Thoracic Surgery, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Search for more papers by this author Dimitrios Boubas Dimitrios Boubas 4th Department of Internal Medicine, Attikon University Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece Search for more papers by this author Justin Stebbing Justin Stebbing orcid.org/0000-0002-1117-6947 Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Sotirios Tsiodras Sotirios Tsiodras 4th Department of Internal Medicine, Attikon University Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece Search for more papers by this author Nadir Arber Corresponding Author Nadir Arber [email protected] orcid.org/0000-0001-5283-6991 The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Author Information Shiran Shapira1,2, Marina Ben Shimon1, Mori Hay-Levi1, Gil Shenberg1, Guy Choshen3, Lian Bannon4, Michael Tepper1, Dina Kazanov1, Jonathan Seni1,2, Shahar Lev-Ari1,2, Michael Peer5, Dimitrios Boubas6, Justin Stebbing7, Sotirios Tsiodras6 and Nadir Arber *,1,2 1The Health Promotion Center and Integrated Cancer Prevention Center, Tel Aviv, Israel 2Department of Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 3Department of Internal Medicine H, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel 4Department of Internal Medicine F, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel 5Department of Thoracic Surgery, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel 64th Department of Internal Medicine, Attikon University Hospital, National and Kapodistrian University of Athens Medical School, Athens, Greece 7Department of Surgery and Cancer, Imperial College, London, UK *Corresponding author. Tel: +972 3 6974968/3716; Fax: +972 3 6974867; E-mail: [email protected] EMBO Mol Med (2022)14:e15997https://doi.org/10.15252/emmm.202215997 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A small but significant proportion of COVID-19 patients develop life-threatening cytokine storm. We have developed a new anti-inflammatory drug, EXO-CD24, a combination of an immune checkpoint (CD24) and a delivery platform (exosomes). CD24 inhibits the NF-kB pathway and the production of cytokines/chemokines. EXO-CD24 discriminates damage-from pathogen-associated molecular patterns (DAMPs and PAMPs) therefore does not interfere with viral clearance. EXO-CD24 was produced and purified from CD24-expressing 293-TREx™ cells. Exosomes displaying murine CD24 (mCD24) were also created. EXO-CD24/mCD24 were characterized and examined, for safety and efficacy, in vitro and in vivo. In a phase Ib/IIa study, 35 patients with moderate–high severity COVID-19 were recruited and given escalating doses, 108–1010, of EXO-CD24 by inhalation, QD, for 5 days. No adverse events related to the drug were observed up to 443–575 days. EXO-CD24 effectively reduced inflammatory markers and cytokine/chemokine, although randomized studies are required. EXO-CD24 may be a treatment strategy to suppress the hyper-inflammatory response in the lungs of COVID-19 patients and further serve as a therapeutic platform for other pulmonary and systemic diseases characterized by cytokine storm. Synopsis In 5% of COVID-19 patients, 5–10 days from disease onset, there is rapid clinical deterioration due to the cytokine storm with no effective therapy. EXO-CD24 is the new immunomodulator with promising efficacy without interfering with pathogen clearance. EXO-CD24 is a novel platform that helps normalize immune activity. EXO-CD24 may represent a new therapeutic opportunity to overcome the devastating effect of COVID-19 and beyond. EXO-CD24 leads to inhibition of tissue injury-driven inflammation without interfering with pathogen-induced immune activation. EXO-CD24 is a targeted innovative technology, based on CD24-enriched exosomes, delivered directly to the lungs to suppress the cytokine storm, and has broad applicability. Introduction Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a newly discovered member of the family of coronaviruses. Most infected individuals are asymptomatic or suffer from mild symptoms including fever, fatigue, and dry cough. In a minority of cases, after several days, clinical deterioration may occur rapidly over a short period of 12–24 h leading to an acute respiratory distress syndrome (Chen et al, 2020). Therapy is required in many of those infected with comorbid diseases (Bhatraju et al, 2020; Goossens et al, 2020; Verity et al, 2020). These patients are prone to develop acute respiratory distress syndrome (ARDS), pulmonary insufficiency, a need for ventilation and even death (Hu et al, 2021). All these severe circumstances are characterized by the cytokine storm (Channappanavar & Perlman, 2017; Fajgenbaum & June, 2020). COVID-19 pathology presents a particular therapeutic challenge as it stems from both rampant viral infection and immune over-reactivity (Channappanavar & Perlman, 2017; Garcia, 2020). Cluster of differentiation (CD) CD24 is a small, heavily glycosylated, membrane-anchored protein which functions as an immune checkpoint regulator (Bradley, 2019; Qiu et al, 2021). CD24 allows immune discrimination between damage-associated molecular patterns (DAMPs) released from damaged or dying cells, and pathogen-associated molecular patterns (PAMPs) derived from pathogens such as bacteria and viruses. The binding of CD24 to DAMPs prevents them from binding to pattern recognition receptors, such as Toll-like receptors (TLRs), and inhibition of DAMP-activation of the nuclear factor-ĸB (NF-ĸB) pathway, a key signaling pathway driving production of cytokines and chemokines (Liu et al, 2009; Barkal et al, 2019). Another distinct class of pattern recognition receptors are Siglecs. CD24 binds both DAMPs and Siglec-10 resulting in activation of the Siglec-10 signaling pathway (Chen et al, 2009; Barkal et al, 2019). This pathway negatively regulates the activity of NF-ĸB, through immunoreceptor tyrosine-based inhibition motif (ITIM) domains associated with SHP-1 (SRC homology 2 (SH2)-domain-containing protein tyrosine phosphatase-1). It is also supported by the identification of a recombinant fusion protein composed of the extracellular domain of CD24 linked to a human immunoglobulin G1 Fc domain as a potential immune checkpoint inhibitor with anti-inflammatory activity (Toubai et al, 2017; Tian et al, 2018). While CD24 dampens DAMPs initiated immune activation, it does not affect PAMPs immune recognition, and hence does not interfere with viral clearance. Exosomes are intraluminal vesicles which play a role in intercellular communication (Colombo et al, 2014; Yáñez-Mó, 2015). Exosomes increase stability and enhance the bioavailability of bioactive compounds (Amreddy et al, 2018; Guo & Jiang, 2020; Elliott & He, 2021). They are in ongoing clinical research as carriers of therapeutic agents against cancer, cardiovascular diseases, diabetesc, graft-versus-host, neurological, and orthopedic diseases (Wolfers et al, 2001; Dai et al, 2008; Le Blanc et al, 2008; Heldring et al, 2015; Newton et al, 2017). A recent study found that exosomes delivered via nebulizer can help repair pulmonary fibrosis-induced lung injury (Dinh et al, 2020). We have developed CD24-enriched exosomes, named EXO-CD24, as a targeted therapy for hyperimmune activation in the context of COVID-19. CD24, which dampens cytokines and chemokines production while not interfering with pathogen clearance, is of particular interest as a therapeutic agent for virus-induced hyper-inflammation and ARDS. EXO-CD24, and its mouse homolog EXO-mCD24, is a new precision nanotechnology that can target and prevent the cytokine storm in the lungs. Herein, the safety and efficacy of EXO-CD24 is shown in vitro, in vivo and in a phase Ib/IIa clinical study. Results EXO-CD24 production and characterization We previously described achieving tightly regulated, tetracycline inducible, overexpression of human CD24 following stable transfection of cloned CD24 into a HEK-293-derived cell-line. We utilized this CD24-stably transfected cell line for the purification of exosomes displaying high levels of CD24. Briefly, CD24 expression was induced by tetracycline for 72 h. Cell growth medium was collected, and CD24-exosomes were isolated using ExoQuick-CG. Isolated exosomes are herein referred to as EXO-CD24. Induced cells demonstrated high expression of EXO-CD24 using both Western blots analysis (Fig 1A) and flow cytometry (Fig 1B). The expression of CD24 in purified exosomes was detected by exosome-based ELISA (Fig 1C) and Western blots which confirmed that CD24 display only on exosomes isolated from cells induced by tetracycline to overexpress CD24 (Fig 1D). The expression of CD24 was also studied using flow cytometry. To allow for size approximation, fluorescent-stained beads of known sizes (100, 200, and 500 nm) were co-analyzed with the EXO-CD24 sample. As expected for known exosomal size, EXO-CD24 were predominantly found in the 100–200 nm size range. In addition, flow cytometry analysis confirmed the copresence of CD24 and exosomal marker CD81 (Fig 1E). EXO-CD24 morphology was studied using cryo-electron microscopy (cryo-EM). The size and morphology of the exosomes with lipid bilayers and vesicular internal structures are clearly visible (Fig 1F). The presence of other exosomal biomarkers such as CD63, CD9, CD81, and HSP70 was detected by several methods (exosome-based ELISA, WB, and flow cytometry) and verified that the isolated population is exosomes. Figure 1. Characterization of CD24-displaying exosomes (EXO-CD24) A. Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 μg/ml tetracycline for 48 h. Twenty microgram from each sample were subjected to Western blot analysis for CD24 and analyzed with anti-CD24 SWA11 mAb. The membrane was reprobed with anti-tubulin antibody to assess the uniformity of sample loading. B. The effect of tetracycline concentration on CD24 expression levels was examined by flow cytometry. C. Exosome-based ELISA was used to measure expression of CD24 in the purified exosomes. D. Western blot analysis of EXO-CD24 exosomes. Tetracycline (Tet) was used to induce CD24 expression in the cell line from which exosomes are derived. HSP70 was used as an exosomal marker. E. Flow cytometry analysis of EXO-CD24. Left panel: Cells were gated based on cell size (forward scatter [FSC] versus side scatter [SSC]); Beads of known sizes of 100, 200 and 500 nm were used for size reference; Middle-left panel: EXO-CD24 labeled with anti-CD24 FITC antibody and gated to the exosome population; Middle-right panel: EXO-CD24 labeled with anti-CD81 APC antibody and gated by to the exosome population; Right panel: EXO-CD24 double labeled with anti-CD24 FITC and anti-CD81 APC antibodies and gated by to the exosome population. F. Cryo-TEM images of isolated CD24-exosomes. On a number of different devices, many experiments were conducted and representative images are shown. Arrows point to double-membraned vesicles (exosomes). Scale bars are 100 nm (left panel) and 200 nm (right panel). Download figure Download PowerPoint In vitro studies EXO-CD24 inhibits PMA-induced cytokine/chemokine secretion in a human monocyte cell line The effect of EXO-CD24 on pro-inflammatory cytokines and chemokines secretion was studied in an in vitro model of human monocytes using the cell line U937. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA), a widely used agent for monocyte/macrophage immune activation. Untreated monocytes grew in suspension showing their known morphological characteristics of small round shaped cells (Fig 2A). PMA-treated cells resulted in their well-characterized phenotype of cell adherence and cell cycle arrest followed by differentiation (Fig 2A). As expected, in comparison with controls, EXO-CD24-treated cells secreted reduced levels of various cytokines and chemokines including IL-1β, RANTES, CD40, IL-1α, IL-6, MCP-1, and MIP-3a/CCL20 (Fig 2B). Figure 2. EXO-CD24 reduces in vitro expression of cytokines and chemokines A. PMA stimulation of monocyte cell line U937 show change in cell morphology and adherence. Untreated (left) and stimulated cells with PMA for 72 h (right). The arrows point to U937 macrophage-like cells (scale bar ×10). B. U937 cells were stimulated with PMA and incubated with (+EXO) or without (−EXO) EXO-CD24 treatment. Incubation medium was then collected and analyzed for cytokines/chemokines levels using a “Multi-plex” array. Data shown are the average of duplicates from a single experiment. Download figure Download PowerPoint To confirm the efficacy and specificity of EXO-CD24 treatment, the exact experiment was repeated, but with the addition of monoclonal antibodies against CD24 that were added simultaneously with EXO-CD24. The effect of the EXO-CD24 was partially blocked by these antibodies. In vivo studies EXO-mCD24 dose toxicity study in mice The safety of EXO-mCD24 administration was examined in a repeated-dose toxicity study in mice. For this purpose, exosomes presenting the murine homolog of CD24 (mCD24) were developed, by transiently induced high mCD24 expression in NIH3T3 murine cell line. A dose of either 5 × 108 or 1 × 109 EXO-mCD24 was administered by inhalation, once daily, for 5 days. Animals were either sacrificed on day 6 or followed for an additional week. Saline, the carrier for the exosomes, was used as vehicle. Administration method (inhalation) and duration (number of days) were designed to mimic the suggested therapeutic use in humans. No adverse effects or differences were observed between control and treated groups in behavior, food and water consumption, body weight, organ weight at the end of the study, nor in hematology, blood chemistry, and urine analyses (Fig 3A–E). In addition, histological evaluation of organs (brain, lung, heart, liver, kidneys, spleen, thymus, and thyroid) from five vehicle-treated and eight animals treated with 1 × 109 EXO-mCD24 showed no effects on organ histology. Figure 3. EXO-CD24 repeated-dose toxicity study in mice, 7-day follow-up A dose of either 5 × 108 and 1 × 109 exosomes/mouse (n = 8 mice/treated group in the main group, n = 3 mice/group in the follow-up groups) was administered by inhalation once daily, for 5 days. Saline, the carrier for the exosomes, was used as vehicle. Animals were followed up during the 5 treatment days and for 7 additional days before being sacrificed. A. Daily body weights (gram). Body weight was measured daily during the first week and three times a week during the second week. Data are average ± SEM. B. Organ weight following a full 5-day treatment course; B: brain, H: heart, Lu: lungs, T: thymus, S: spleen, K: kidneys, Li: liver. Data are average ± SEM. C. Urine analysis. The urine was collected and analyzed and no abnormalities were found. D. Hematology. Mice were anesthetized and blood was taken for full hematology tests. Data are average ± SEM. E. Blood Chemistry. Mice were anesthetized and blood was taken for clinical chemistry tests. Data are average ± SEM. Data information: Data are expressed as mean ± SEM; vehicle group n = 5 mice; n = 8 mice per treatment group. Raw values and statistical analyses are provided in Appendix Supplementary Methods. Download figure Download PowerPoint EXO-mCD24 reduce cytokine release and lung inflammatory reaction in an ARDS model Next, we investigated the efficacy of mCD24-exosomes in LPS-induced ARDS mouse model. Exosomes presenting the murine homolog of CD24 (mCD24) were developed, by transiently induced high HSA expression (Fig 4A–C) in Expi293 embryonic cell line. Figure 4. Characterization of mCD24-displaying exosomes (EXO-mCD24) A. Exosome-based ELISA was used to measure expression of mCD24 in the purified exosomes. The expression level was compared to mCD24-negative exosomes and to mouse recombinant protein. The data are represented as average of three technical replicates ± SEM. B. Flow cytometry analysis of EXO-mCD24. EXO-mCD24 labeled with APC-conjugated anti-mCD24 antibody and gated to the exosome population. C. Western blot analysis of EXO-mCD24 exosomes. HSP70 was used as an exosomal marker. Download figure Download PowerPoint The animals were challenged by intratracheal introduction of LPS, followed by mCD24 exosome treatment, at 1 × 108 or 1 × 109 EXO-mCD24, once daily for 3 days and were then sacrificed (Fig 5A–C; n = 10 per group). Naïve mice (not challenged by LPS) served as overall control (n = 5) and saline-treated mice used as vehicle control treatment (n = 10). As expected, histology confirm normal healthy lungs in the naïve animals, while the lungs in all LPS-challenged groups had a multifocal coalescing inflammatory reaction, composed predominantly of neutrophils. The inflammatory infiltrates were mainly perivascular but also observed around the middle sized and small bronchiole (Fig 5). Animals treated with saline or low-dose EXO-mCD24 (1 × 108) had moderate-to-severe lung injury with a score of 4.7 ± 1.1 and 4.6 ± 0.8, respectively. Animals in the high-dose treatment group (1 × 109 EXO-mCD24) presented a moderate lung injury, with a score of 4.0 ± 0.8. A marked reduction in cytokine and chemokine levels (IL-6, IL-12, TNF-α, IFN-γ, and KC/CXCL1) was observed, in animals treated with 1 × 109 EXO-mCD24 in the sera (Fig 5, upper panel) and in the BAL (Fig 5, lower panel), most of them in a dose-depended manner. These results suggest that treatment with CD24-exosomes may curtail inflammation. Figure 5. EXO-mCD24 attenuates lung injury in a mice ARDS model A. Study design. Animals were challenged by intratracheal introduction of LPS, followed by EXO-HSA treatment, at 1 × 108 or 1 × 109 EXO-HSA/mouse, once daily for 3 days and then, mice were sacrificed. Serum and BAL fluid (Bronchial alveolar lavage fluid) were collected. B. Histopathology. Representative histological images of lung tissue. Tissues from saline and 1 × 108 EXO-HSA/mouse (low dose) treatment show extensive neutrophil infiltrate in the alveolar spaces (arrows) and around the bronchi and blood vessels (small arrows). In comparison, the inflammatory infiltrate in the 1 × 109 EXO-HSA/mouse (high dose) treatment is considerably attenuated. All images: hematoxylin and eosin (H&E) staining (naïve: no LPS challenge). C. The levels of systemic and local cytokines and chemokines. Serum (upper panel) and BAL fluid (lower panel) cytokines/chemokines levels. (KC/CXCL1, a neutrophil chemokine). Data are average ± SEM, n = 10 mice/group. Download figure Download PowerPoint Inhaled EXO-mCD24 increase survival in a mouse sepsis model EXO-mCD24 treatment was examined in a cecum ligation and puncture (CLP)-induced sepsis model in mice. The animals underwent the CLP procedure followed by vehicle- or EXO- mCD24-treatment, at 1 × 109/1 × 1010/1 × 1011 mCD24-exosomes/mouse, by inhalation at 1-, 8-, and 24-h post-surgery. Analysis of serum cytokine levels did not show differences between the vehicle and treatment groups. However, the control group had higher mortality compared with the group treated with 1 × 1010 EXO- mCD24 (Log Rank: P < 0.001) or 1 × 1011 EXO-mCD24 (Log Rank: P = 0.001; Fig 6A). Cox regression showed longer survival in the 1 × 1010 EXO-mCD24group (hazard ratio = 0.069; 95% CI, 0.016–0.292) and in the 1 × 1011 exosomes/mouse group (hazard ratio = 0.155; 95% CI, 0.043–0.555) compared with the control group. The mortality rate and survival were comparable in the 1 × 109 EXO-mCD24 and the control group (hazard ratio = 0.684; 95% CI, 0.255–1.838). Figure 6. In vivo PK of EXO-HSA and its efficacy in a mice sepsis model A. Cecum ligation and puncture (CLP) was performed, followed by inhalation treatment with vehicle, or 1 × 109, 1 × 1010, 1 × 1011 EXO HSA/mouse, at 1, 8 and 24 h post-surgery. B, C. EXO-CD24 was isolated from BALF (B) and serum (C) of treated and control mice. Evaluation of CD24/CD81-positive exosomes, at each time point, was carried out by flow cytometry. The data are presented as average (n = 4 mice at each time point) ± SEM. Download figure Download PowerPoint Pharmacokinetic study The pharmacokinetic and pharmacodynamics parameters had been evaluated, following a single inhalation of EXO-CD24. It was detected in the BALF (Fig 6B) and even in the bloodstream (Fig 6C), without accumulation in any organ. Phase Ib/IIa clinical study Thirty-five patients were enrolled, between September 26th, 2020, and February 13th, 2021, in four dose escalation groups (Fig 7A). Figure 7. Enrollment and clinical results A. One trial participant, in the 1 × 109 EXO-CD24/dose group discontinued the treatment after 3 days due to sustained low blood oxygen saturation. B, C. (B) respiratory rate and (C) Blood oxygen (SpO2) and before treatment (Day 1, measured before administration of first dose) and after treatment (Day 7). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for Blood oxygen (SpO2) and respiratory rate. These are presented graphically via box plots before and after treatment. D. Representative X-ray image. Download figure Download PowerPoint Group 1: five patients that were enrolled one by one with a waiting time of at least 2 weeks between patients. They received EXO-CD24 at a concentration of 1 × 108 particles per 2 ml saline solution. Safety findings for each individual patient of the first group were reported to the hospital's IRB, and following approval by the IMOH, the next patient group was recruited. Subsequent groups were enrolled following a review of the safety data of the preceding group by the IMOH. Group 2: A dose escalation group of five patients received EXO-CD24 at a concentration of 5 × 108 particles per 2 ml saline solution. Following IMOH approval, the third group was recruited. Group 3: 20 patients received 1 × 109 exosomes. The drug seemed to be very safe with possible efficacy. The IMOH granted recruitment of Group 4: five patients that received 1 × 1010 exosomes. Participants were free to withdraw from participation in the study at any time. All patients, except for one, completed the full treatment regimen of five inhalations and completed the study as planned. The overall eligible study population was mostly comprised of men (65.7%). The mean ± SD participant age at baseline was 57.5 ± 11.46 (range 33–77) years. The mean time from COVID-19 diagnosis until the first treatment was 9.5 days. The mean, upon enrollment, blood oxygen saturation (SpO2), and respiratory rate were 90.7% (range 90–94) and 27.6 (range 23–30) breaths/min, respectively. Average CRP prior to treatment was 127.0 ± 14.7 (SEM) mg/l. Thirty-two of the participants were white, one black, one Latin American, and one Asian. Safety All adverse effects were classified as unrelated or probably not related to EXO-CD24 (Table 1). During the study period, 42 AEs irrespective of causality to the investigational product were reported for 29 (82.9%) patients. The AEs noted in ≥ 10% of the patients by MedDRA System Organ Class were investigations (11 events in 9 [25.71%] patients), followed in descending frequency order by blood and lymphatic system (6 events in 6 [17.14%] patients), nervous system (6 events in 5 [14.29%] patients), and general disorders (5 events in 5 [14.29%] patients). The only AE by MedDRA Preferred Term noted in ≥ 10% of the patients was leukocytosis (11.43%) related to the steroid therapy. The only reported SAE occurred in a patient enrolled in Group 1 (Patient 003). It was an acute asthmatic exacerbation in a patient with asthma. The event occurred toward the end of the follow-up period and improved dramatically, as expected, with the administration of ventolin and steroids inhalers. Another patient, who suffers from chronic lymphocytic leukemia and was immunocompromised due to rituximab therapy, completed the trial successfully with viral eradica
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