LARGE SCALE MANUFACTURING STRATEGY FOR PRODUCTION OF UMBILICAL CORD-DERIVED MESENCHYMAL STEM CELLS IN SUPPORT OF CLINICAL TRIALS

Background & Aim: Mesenchymal stem cells (MSCs) have been shown to modulate hyperinflammation, promote tissue repair and secrete antimicrobial factors. MSCs have been studied in clinical trials of autoimmune diseases, inflammatory disorders, refractory GvHD and acute respiratory distress syndrome (ARDS). MSCs can be isolated and expanded from multiple tissues, including umbilical cord (UC). A number of clinical studies demonstrated safety and feasibility of UCMSCs therapy for the treatment of COVID-19 ARDS. UC-derived MSCs are easily available and can be quickly expanded to relevant numbers. UC-MSCs have an extended population doubling capacity and express low levels of class I and class II leukocyte antigen, which may reduce alloreactivity. To meet clinical manufacture demands, UC-MSC production requires an innovative, scaled-up manufacturing platform. We describe the manufacturing strategy developed in support of a double-blind, randomized, controlled UC- MSC clinical trial in subjects with COVID-19 ARDS. Methods, Results & Conclusion: UC-MSC Final Product was manufactured from the master cell bank (MCB) derived from subepithelial lining of a UC from a healthy term delivery, in cGMP conditions. Utilizing a 2D culture xenogeneic protein-free process, UC-MSC MCB was culture-expanded during 3 expansion cycles, in tissue culture treated vessels with increased surface area for each expansion, in commercially available tissue culture media supplemented with platelet lysate. Cells were harvested during log phase, at 75-80% confluence. The manufacturing process yielded ~ 300x increase in total viable cells at the end of the last expansion cycle. The Final Product was cryopreserved using a controlled rate freezer. Each subject in the treatment group received two doses of 100×106 UC-MSCs. A single UC-MSC Final Product batch was sufficient to treat all subjects randomized to the treatment group and complete the trial. The final product was tested for identity (label verification), effectiveness by viable cell dose and cell viability (>80%), safety by assessment of endotoxin (<1.65 EU/ml), Mycoplasma (negative), 14-day Sterility (negative) and purity by [Figure presented] FLOW cytometry (CD90/CD105?>90%, CD34/CD45 <10%). UC-MSC cell doses prepared for infusion produced similar results to UC-MSC Final Product when tested to confirm product identity, effectiveness, safety and purity. The developed 2D culture and expansion process can be successfully scaled up without compromising integrity of the final UC-MSC product.

Umbilical Cord-derived Mesenchymal Stem Cells modulate TNF and soluble TNF Receptor 2 (sTNFR2) in COVID-19 ARDS patients.

OBJECTIVE: We aimed at explaining the mechanism of therapeutic effect of Umbilical Cord Mesenchymal Stem Cells (UC-MSC) in subjects with COVID-19 Acute Respiratory Distress Syndrome (ARDS). Patients with COVID-19 ARDS present with a hyperinflammatory response characterized by high levels of circulating pro-inflammatory mediators, including tumor necrosis factor α and ß (TNFα and TNFß). Inflammatory functions of these TNFs can be inhibited by soluble TNF Receptor 2 (sTNFR2). In patients with COVID-19 ARDS, UC-MSC appear to impart a robust anti-inflammatory effect, and treatment is associated with remarkable clinical improvements. We investigated the levels of TNFα, TNFß and sTNFR2 in blood plasma samples collected from subjects with COVID-19 ARDS enrolled in our trial of UC-MSC treatment. PATIENTS AND METHODS: We analyzed plasma samples from subjects with COVID-19 ARDS (n=24) enrolled in a Phase 1/2a randomized controlled trial of UC-MSC treatment. Plasma samples were obtained at Day 0 (baseline, before UC-MSC or control infusion), and Day 6 post infusion. Plasma concentrations of sTNFR2, TNFα, and TNFß were evaluated using a quantitative multiplex protein array. RESULTS: Our data indicate that at Day 6 after infusion, UC-MSC recipients develop significantly increased levels of plasma sTNFR2 and significantly decreased levels of TNFα and TNFß, compared to controls. CONCLUSIONS: These observations suggest that sTNFR2 plays a mechanistic role in mediating UC-MSC effect on TNFα and TNFß plasma levels, determining a decrease in inflammation in COVID-19 ARDS.

Umbilical Cord-derived Mesenchymal Stem Cells for COVID-19 Patients with Acute Respiratory Distress Syndrome (ARDS).

The coronavirus SARS-CoV-2 is cause of a global pandemic of a pneumonia-like disease termed Coronavirus Disease 2019 (COVID-19). COVID-19 presents a high mortality rate, estimated at 3.4%. More than 1 out of 4 hospitalized COVID-19 patients require admission to an Intensive Care Unit (ICU) for respiratory support, and a large proportion of these ICU-COVID-19 patients, between 17% and 46%, have died. In these patients COVID-19 infection causes an inflammatory response in the lungs that can progress to inflammation with cytokine storm, Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS), thromboembolic events, disseminated intravascular coagulation, organ failure, and death. Mesenchymal Stem Cells (MSCs) are potent immunomodulatory cells that recognize sites of injury, limit effector T cell reactions, and positively modulate regulatory cell populations. MSCs also stimulate local tissue regeneration via paracrine effects inducing angiogenic, anti-fibrotic and remodeling responses. MSCs can be derived in large number from the Umbilical Cord (UC). UC-MSCs, utilized in the allogeneic setting, have demonstrated safety and efficacy in clinical trials for a number of disease conditions including inflammatory and immune-based diseases. UC-MSCs have been shown to inhibit inflammation and fibrosis in the lungs and have been utilized to treat patients with severe COVID-19 in pilot, uncontrolled clinical trials, that reported promising results. UC-MSCs processed at our facility have been authorized by the FDA for clinical trials in patients with an Alzheimer's Disease, and in patients with Type 1 Diabetes (T1D). We hypothesize that UC-MSC will also exert beneficial therapeutic effects in COVID-19 patients with cytokine storm and ARDS. We propose an early phase controlled, randomized clinical trial in COVID-19 patients with ALI/ARDS. Subjects in the treatment group will be treated with two doses of UC-MSC (l00 × 106 cells). The first dose will be infused within 24 hours following study enrollment. A second dose will be administered 72 ± 6 hours after the first infusion. Subject in the control group will receive infusion of vehicle (DPBS supplemented with 1% HSA and 70 U/kg unfractionated Heparin, delivered IV) following the same timeline. Subjects will be evaluated daily during the first 6 days, then at 14, 28, 60, and 90 days following enrollment (see Schedule of Assessment for time window details). Safety will be determined by adverse events (AEs) and serious adverse events (SAEs) during the follow-up period. Efficacy will be defined by clinical outcomes, as well as a variety of pulmonary, biochemical and immunological tests. Success of the current study will provide a framework for larger controlled, randomized clinical trials and a means of accelerating a possible solution for this urgent but unmet medical need. The proposed early phase clinical trial will be performed at the University of Miami (UM), in the facilities of the Diabetes Research Institute (DRI), UHealth Intensive Care Unit (ICU) and the Clinical Translational Research Site (CTRS) at the University of Miami Miller School of Medicine and at the Jackson Memorial Hospital (JMH).