Efficacy of intravenous infusions of UC-derived MSCs for the treatment of 1 COVID-19: A structured summary of a phase II double blinded, randomized 2 controlled clinical trial

45 In this clinical research project, we will conduct a highly rigorous clinical trial where we randomize the 46 patients between umbilical cord (UC)-derived mesenchymal stem cell (MSC) and standard of care 47 (SOC). This study will ask the question whether the allogenic MSCs are effective in reducing death 48 and/or progression of COVID-19 disease when administered to patients with moderate to severe 49 symptoms. MSCs are unique group of cells with minimal immune reactivity and produce multiple 50 beneficial effects, which include reduction of severe inflammatory reaction. There is convincing 51 evidence from human studies that intravenous (IV) delivery of MSCs can directly impact the hyper- 52 inflammatory responses of COVID-19 induced injury of the heart and lungs. Our study will evaluate the 53 safety and efficacy of allogenic UC-derived MSCs administered intravenously to COVID-19 patients 54 with moderate to severe symptom in a randomized, controlled clinical trial. In addition, we will 55 determine whether : (i) UC-derived MSCs are effective in reducing 30-day all-cause mortality (primary 56 endpoint) and (ii) UC-derived MSCs are effective in preventing progression to mechanical ventilation, 57 and/or reducing an inflammatory response, and/or improving overall patient condition (secondary 58 endpoints). 59

from SARS-CoV-2 family of viruses can cause illnesses such as common cold, severe acute respiratory 74 syndrome (SARS), and Middle East Respiratory Syndrome (MERS). Coronavirus is an enveloped 75 single-stranded RNA virus that causes distress in respiratory, enteric, hepatic and neurological systems 76 with varying severity among human (1)(2). The main symptoms of COVID-19 include fever, fatigue, and 77 cough that can progress rapidly to severe and critical conditions resulting in pulmonary edema leading 78 to acute lung injury (ALI) and acute respiratory distress syndromes (ARDS) (3-7). 79 In the last eight months or so, it has spread to 188 countries and as of October 14, 2020, it has infected 80 more than 38 million people with 1,083,234 deaths worldwide (8). Importantly, there are currently no 81 approved drugs or vaccines for the treatment of COVID-19 patients. The clinical spectrum of COVID-19 82 stems from over-activated immune system of the infected subjects, to kill the virus, which can 83 induce/stimulate/ give rise to the production of a large number of inflammatory factors, resulting in a 84 severe "cytokine storm". The cytokine storm may induce organ damage followed by edema, dysfunction 85 of air exchange, acute respiratory distress, acute cardiac injury, and secondary infection, which may 86 lead to death. 87 Stem cell therapy has emerged as a revolutionary treatment for diseases that were considered 88 untreatable only a few years ago. Umbilical cord (UC)-derived mesenchymal stem cells (MSCs) have 89 been shown to repair damaged liver, kidney, heart, pancreas, skin, cartilage, and cornea in animal 90 Design and study plan 113 The phase II study aims to recruit 20 subjects to assess the safety and efficacy of UC-derived MSCs for 114 treatment of COVID-19 versus standard of care (SOC) during 30 day follow up. It is two arms 115 randomized study. Subjects will be divided in the following 2 groups: 116 Group 1: Fifteen subjects will be treated with three intravenous infusions of 5 x 10 5 (per kg body 117 weight) UC-derived MSCs delivered via peripheral intravenous infusion on days 1, 3, and 5 besides the 118 standard of care (SOC). 119 Group 2: Five subjects will be treated under SOC. Two subjects in group 1 will not be treated 24 hours 120 apart. Patients will be randomized in a 3:1 ratio (UC-derived MSCs : SOC). 121 We propose to use this intervention at the onset of moderate to severe COVID-19. Figure 1  • SOFA Score. Each organ system is assigned a value for 0 (normal) to 4 (highest degree of 130 dysfunction) 131 • Rate of all-cause mortality (measured at day 30 or at hospital discharge, whichever comes first) 132 • Clinical respiratory changes 133 • Chest imaging changes for 30 days 134 • Complete blood count (CBC) with differential 135 • Comprehensive metabolic panel 136 • CD4 + and CD8 + cell count 137 • Interleukin (IL)-10 levels 138 • Tumor necrosis factor (TNF)-α levels 139 • C-reactive protein levels 140 • Lactate dehydrogenase levels 141 • D-dimer levels 142 • Ferritin levels 143 • pro-type B natriuretic peptide (pro-BNP) levels 144 • Improvement of clinical symptoms including duration of fever and respiratory efficacy of 145 treatment 146

Isolation and characterization of human UC-derived MSCs 147
Human umbilical cord tissue along with informed consent forms will be collected from hepatitis B, C, 148 and COVID-19 virus-negative women with healthy pregnancies during the Cesarean Section surgery 149 after completion of normal gestation period. The cord tissue will be transported in sterile phosphate-150 buffered saline (PBS) containing 200 units/ml penicillin and 200 µg/ml streptomycin on ice. In the 151 biosafety cabinet, the cord will be washed with 4-5 changes of sterile 1x PBS and placed in a Petri plate 152 with 15 ml PBS. Next, the umbilical cord tissue will be minced after removal of blood vessels. Minced 153 pieces will be treated with collagenase type-I (201 U/ml collagenase type I) for ~3.5 hours. Following 154 centrifugation of digested lysate diluted with PBS, the cells will be seeded in tissue culture flasks and 155 will be placed in an incubator at 5% CO2, 95% humidity at 37⁰C. The cells will be fed with fresh media 156 comprising of Dulbecco's modified Eagle's medium (DMEM)-high glucose supplemented with 20% 157 Hyclone fetal bovine serum (FBS), 1% 10,000 U/mL penicillin, and 10,000 μg/mL streptomycin every 158 third day. UC-derived MSCs at passage 3 (P3) will be characterized at the time of cryopreservation 159 using the immunocytochemical analysis to determine the expression of positive surface markers i.e. 160 CD73, CD90, C105, and negative for CD34 and CD45. In addition, quality control analyses will include 161 sterility analysis for any signs of bacterial and fungal contamination. The culture-expanded cells will be 162 cryopreserved at P3 using standard cryopreservation protocol until used in the experiment.

UC-derived MSC production facility 164
Cell production will be performed in a designated clean room qualified to maintain air quality of ISO-165 9001: 2015 standards. All tissue handling and final product filling will be completed within a certified 166 Class II Biological Safety Cabinet. Furthermore, all members of staff are required to gown-in to the 167 clean room space with sterile, single-use personal protective gear. Clean room, certified to meet ISO-168 9001: 2015 standards, will be utilized for processing activities. The Facility has an established system 169 for sanitization and disinfection of clean-room, and the equipment used in manufacturing processes, in 170 order to produce aseptic conditions. 171 The organization maintains a quality enforcement program to ensure that each product supplied 172 conforms to product specifications. Product release will be monitored by trained staff who has 173 appropriate training and experience in evaluating and determining product quality. It will be ensured 174 that personnel engaged in technical and scientific activities have the necessary technical and scientific 175 education, training, background and experience to ensure competent performance of their assigned 176

Infusion of UC-derived MSCs in COVID-19 patients 178
The stem cell preparation will be administered intravenously in three single infusions of 5 x 10 5 cells per 179 kilogram body weight each on days 1, 3, and 5 in addition to the SOC. For the intravenous infusion, 180 stem cells will be resuspended in 100 ml of normal saline (0.9% sodium chloride). The cell infusion will 181 be performed for approximately 50 minutes with a speed of about 35 drops per minute. The patient will 182 be critically assessed by the clinician during infusion as well as six hours post-infusion for infusional 183 and allergic reactions. The laboratory, clinical, and radiological outcomes will be recorded according to 184 parameters mentioned in Table 1. 185

Time Frame 186
All patients will be examined at the following time points:

Statistical methods 196
All statistical comparisons will be made primarily between the baseline and final values using the 197 Student's paired t-test. Results will be obtained as means and standard deviations. The p value <0.05 198 will be considered statistically significant. A 95% confidence interval for the difference in means will be 199 used for double confirmation. 200

Patient recruitment criteria 201
The study will recruit twenty patients suffering from COVID-19 with moderately severe symptoms. To 202 participate in the proposed study, the patients must meet the following inclusion and exclusion criteria: 203

Inclusion criteria: 204
For inclusion, the subjects must comply with the following criteria: 205 • Provide written informed consent. with worsening hypoxia compatible with mild ARDS (PaO2/FiO2 </= 300 but > 200). 215 • Appropriate venous access. 216 • Female patients will be agreed to use recommended birth control until 6 months post-treatment. 217 • Must agree to conform with all study constraints including completing all study visits. 218 • Entails in-patient admission. 219

Exclusion: 220
The following exclusion criteria will be used: 221 • Male or female younger than 18 years of age. 222 • Subjects with mild COVID-19 symptoms. Mild disease category symptoms include fever, cough, 223 headache, myalgia, sore throat, nasal congestion, nausea, vomiting, diarrhea, fatigue, anosmia 224 and/or dysgeusia. 225 • A female who is pregnant, nursing the infant, or of child-carrying potential while not practicing 226 effective contraceptive methods. Females to be included in the present study must undergo a 227 blood pregnancy test at screening and before infusion. 228 • Failure to perform any of the evaluations necessary for endpoint analysis. 229 • Have a clinical history of melanoma within 3 years, i.e. subjects with prior malignancy must be 230 disease-free for 3 years. 231 • History of drug abuse (illegal drugs or prescription medications not being used properly for a 232 pre-existing medical condition) or alcohol abuse or documented medical or legal problems 233 arising from the use of alcohol or drugs within the past 2 years. 234 • If found serum positive for HIV, hepatitis B or C. 235

Events/visits schedule 236
Treatment evaluation will be performed through study design including a schedule of event/visit follow-237 up as described below. It is a two arms study to evaluate the safety and potential efficacy of UC-derived 238

MSC infusions for the treatment of COVID-19 infection. 239
Visit 1: Screening, written consent, duration of fever and respiratory efficacy, chest imaging, 240 quantitative real-time PCR, ELISA, CBC with differential, comprehensive metabolic panel, SOFA score, 241 CD4 + and CD8 + cell count, IL-10 levels, TNF-α levels, C-reactive protein levels, D-dimer levels, lactate 242 dehydrogenase levels, ferritin levels, and pro-BNP levels will be evaluated. imaging, quantitative real-time PCR, ELISA, CBC with differential, comprehensive metabolic panel, 259 SOFA score, CD4 + and CD8 + cell count, IL-10 levels, TNF-α levels, C-reactive protein levels, D-dimer 260 levels, lactate dehydrogenase levels, ferritin levels, and pro-BNP levels will be examined. Needle Insertion for IV Infusion: An experienced physician will perform this procedure. There may be 269 discomfort due to needle insertion lasting a few minutes. Occasionally, a small accumulation of blood 270 Steps taken to minimize the risks: The UC-derived MSCs undergo extensive screening and in-process 279 controls to ensure purity and consistency during manufacturing. 280

Scheme of assessment: 262
Intravenous administration of study product, as well as blood draws will only be performed by qualified, 281 licensed, medical experts. Although unanticipated, there is a hypothetical risk of thrombus formation 282 associated with administration of any biologic product. Should the patient experience symptoms of 283 thrombosis, based on the physician's judgment, the patient will be treated for thromboembolisms as per 284 ICU protocol. 285 286 287