Novel Renal Autologous Cell Therapy for Type 2 Diabetes Mellitus Chronic Diabetic Kidney Disease: Clinical Trial Design

<b><i>Background:</i></b> Cell therapies explore unmet clinical needs of patients with chronic kidney disease with the potential to alter the pathway toward end-stage kidney disease. We describe the design and baseline patient characteristics of a phase II multicenter clinical trial utilizing the novel renal autologous cell therapy (REACT), by direct kidney parenchymal injection via the percutaneous approach in adults with type 2 diabetic kidney disease (T2DKD), to delay or potentially avoid renal replacement therapy. <b><i>Design:</i></b> The study conducted a prospective, multicenter, randomized control, open-label, phase II clinical trial between an active treatment group (ATG) receiving REACT from the beginning of the trial and a contemporaneous deferred treatment group (DTG) receiving standard of care for 12 months before crossing over to receive REACT. <b><i>Objectives:</i></b> The objective of this study was to establish the safety and efficacy of 2 REACT injections with computed tomography guidance, into the renal cortex of patients with T2DKD administered 6 months apart, and to compare the longitudinal change in renal function between the ATG and the DTG. <b><i>Setting:</i></b> This was a multicenter study conducted in major US hospitals. <b><i>Patients:</i></b> We enrolled eighty-three adult patients with T2DKD, who have estimated glomerular filtration rates (eGFRs) between 20 and 50 mL/min/1.73 m². <b><i>Methods:</i></b> All patients undergo an image-guided percutaneous kidney biopsy to obtain epithelial phenotype selective renal cells isolated from the kidney tissue that is then expanded ex vivo over 4–6 weeks, resulting in the REACT biologic product. Patients are randomized 1:1 into the ATG or the DTG. Primary efficacy endpoints for both study groups include eGFR measurements at baseline and at 3-month intervals, through 24 months after the last REACT injection. Safety analyses include biopsy-related complications, REACT injection, and cellular-related adverse events. The study utilizes Good Clinical and Manufacturing Practices and a Data and Safety Monitoring Board. The sample size confers a statistical power of 80% to detect an eGFR change in the ATG compared to the DTG at 24 months with an α = 0.05. <b><i>Limitations:</i></b> Blinding cannot occur due to the intent to treat procedure, biopsy in both groups, and open trial design. <b><i>Conclusion:</i></b> This multicenter phase II randomized clinical trial is designed to determine the efficacy and safety of REACT in improving or stabilizing renal function among patients with T2DKD stages 3a–4.

Deterministic Cell Separation Recovers >2-Fold T Cells, and More Naïve T Cells, for Autologous Cell Therapy As Compared to Centrifugally Prepared Cells

CAR-T autologous cell therapies are delivering impressive results in the clinic. However, there are still significant manufacturing challenges impeding the rapid adoption of these advanced therapies. On the first day of cell processing, most manufacturing approaches require ~5 steps (~4 hours) to obtain a white blood cell (WBC) preparation sufficiently depleted of red blood cells (RBCs) for T-cell selection and activation steps; and involves large cell losses and a great deal of inconsistency. Here we present a single-step procedure that yields &gt;2 fold more cells that centrifugal processing with comparable or better quality in &lt;1 hour. We previously reported a small-scale microfluidic approach using deterministic cell separation (DCS) to effectively isolate and separate WBCs with high recoveries, no loss of WBC subtypes, no cell damage, and greater numbers of central memory T cells than traditional Ficoll-based processing. Extending this work, we now present the results of our fully scaled-up processing of 23 normal donor leukopaks and 4 disease samples using a full-scale DCS prototype. All samples were processed in &lt;45 minutes, with only an additional 10 minutes hands-on time. On average, inclusive of aggregate removal by prefiltering, DCS achieved 88% WBC recovery, 94% RBC removal, and 98% platelet ( PLT) removal from the undiluted leukopak samples (n=23). Furthermore, DCS resulted in a RBC/WBC ratio of 0.1 compared with a ratio of 1.4 for Ficoll. Similarly, the PLT/WBC ratios were 0.89 versus 7.17 for DCS and Ficoll, respectively (n=20). In addition, DCS preparations contained 2-fold more CD3+ T cells (n=17), and, importantly, the CD4+ cells were less differentiated (more cells in naïve and central memory stages) than those recovered by Ficoll. Similarly, DCS processed blood from cancer patients had a ratio of RBC/WBC = 7.0 versus 20.1 for Ficoll, and a PLT/WBC ratio = 0.7 versus 15.6 for Ficoll (n=4). These results demonstrate the capabilities of DCS in processing not only samples from normal donors but also blood from cancer patients with similar efficiencies. Further, with DCS we achieved wash efficiencies of more than 3 log, without the typically associated cell loss, as demonstrated by the removal of viral particles, soluble proteins and cytokines and growth factors present in plasma. Therefore, cells from leukopaks processed by DCS can be washed and collected directly into cell culture media, or other solutions, to ready them for downstream applications without pelleting and repeated washes, greatly simplifying workflows. We integrated our DCS technology into a full scale parallelized, disposable, closed fluid path solution and automated platform prototype, the Curate ® Cell Processing System, capable of processing undiluted leukopacks at rates in excess of 300mL/hour. Designed to process blood products in bags using a single-use cassette containing microfluidic components, the Curate ® delivers a debulked WBC product to a bag. With a hands-on time of only 10 minutes, the Curate ® reduces the time to activation- and expansion-ready cells from leukopaks by 6-fold as compared with centrifugation and elutriation methods (Bowles, et al. Cytotherapy 2018;20(5):S109). The system can process a full leukopak (200-300 mL containing up to 1.2x10 10 WBC) within 40 minutes with a maximal cell throughput of 1.8x10 10 WBC per hour. Additionally, the same Curate ® device can be used to achieve up to 200x10 6 cell/mL in as little as 40 mL of media and without requiring pelleting. In summary, we believe our technology enables a significant breakthrough in the production of CAR-T cells by efficiently recovering more and cleaner total and naÏve T cells, for CAR-T cell production. Furthermore, the closed-system Curate ® will simplify cell processing workflows by reducing the number of cell washing steps, as well as the hands-on time and resources. Supported in part by NIH Grant No 5R42CA228616-03 Disclosures Behmardi: GPB Scientific, Inc: Current Employment. Ouaguia: GPB Scientific, Inc: Current Employment. Healey: GPB Scientific, Inc: Current Employment. Jones: GPB Scientific, Inc: Current Employment. Rahmo: GPB Scientific, Inc: Current Employment. Skelley: GPB Scientific, Inc: Current Employment. Gandhi: GPB Scientific, Inc: Current Employment. Campos-Gonzalez: GPB Scientific, Inc: Current Employment, Current holder of stock options in a privately-held company. Civin: GPB Scientific, Inc: Current holder of individual stocks in a privately-held company. Ward: GPB Scientific, Inc: Current Employment.

A Comparison of Peripheral Blood Smears, Autologous Cell Cultures, and Reverse Line Blot Hybridisation in Screening for Anaplasma/Ehrlichia in Roaming Dogs and Symptomatic Dogs in Trinidad

This study compared two methods to detect cases of canine ehrlichiosis in a field setting. One method was a polymerase chain reaction for the 16S rRNA gene followed by reverse line blot hybridisation with genera and species-specific probes for Anaplasma/Ehrlichia. The second method was an autologous cell culture of peripheral leucocytes isolated from heparinised blood and maintained in a homologous canine serum in Dulbecco’s Modified Eagle medium without antibiotics. The cultures were examined under light microscopy for inclusion bodies after 48 h. Leucocytes were successfully propagated for 20 of the 34 samples submitted for autologous cell culture. Inclusion bodies were observed after cell culture in leucocytes of eight dogs. Two dogs were positive to the Anaplasma/Ehrlichia genera probe and six dogs were positive to the E. canis probe after reverse line blot hybridisation. There was acceptable agreement between reverse line blot hybridisation and cell culture results. Both reverse line blot hybridisation and autologous cell cultures can be used to detect E. canis in subclinical and clinical cases of disease. A definitive diagnosis of E. canis is best achieved by a combination of clinical signs, positive autologous cell culture, and reverse line blot hybridisation results.

A Canadian perception of the nursing practice, research, and theoretical implications associated with Autologous Cell Transplantation

Heart failure is a progressive disorder. An estimated 400,000 Canadians are diagnosed annually with heart failure, and a quarter experience severe heart failure that is unresponsive to medical therapy. Autologous cell transplantation (ACT) has been proposed as a new approach for cardiac repair, and holds enormous potential for the regeneration of injured myocardium cells. Currently, ACT is under investigation in Canada. The use of ACT as a treatment alternative for heart failure patients has been established over the past 5 years across Europe and the United States. This paper will present a Canadian perception of the nursing practice, research, and theoretical implications associated with this new and innovative therapy.

Impact of endoscopic submucosal dissection and epithelial cell sheet engraftment on systemic cytokine dynamics in patients with oesophageal cancer

The tumour microenvironment (TME) plays an important role in cancer development, progression, and metastasis. Various cytokines are present in the TME in oesophageal cancer. Oesophageal stricture is a major complication of endoscopic submucosal dissection (ESD) for oesophageal cancer, and inflammatory cytokines are closely related to its pathogenesis. However, the cytokine crosstalk involved in the oesophageal cancer TME and post-ESD stricture has not been fully elucidated. This study investigated the comprehensive cytokine dynamics following ESD in patients with oesophageal cancer. In addition, the effect of a novel preventive technique for post-ESD stricture, autologous cell sheet engraftment, on cytokine levels was evaluated. Various pro-inflammatory and anti-tumorigenic cytokines were elevated in patients with oesophageal cancer, and ESD transiently influenced cytokine concentrations. IL-1β and TNF-α, two major pro-inflammatory cytokines that induce oesophageal stricture, were significantly suppressed by cell sheet engraftment. In conclusion, this study revealed the distinct cytokine dynamics after ESD in patients with oesophageal cancer, together with the effect of autologous cell sheet engraftment on cytokine fluctuation. These results can accelerate research on the TME and therapeutic strategies for oesophageal cancer.