MSC Manufacturing for Academic Clinical Trials: From a Clinical-Grade to a Full GMP-Compliant Process

Following European regulation 1394/2007, mesenchymal stromal cell (MSCs) have become an advanced therapy medicinal product (ATMP) that must be produced following the good manufacturing practice (GMP) standards. We describe the upgrade of our existing clinical-grade MSC manufacturing process to obtain GMP certification. Staff organization, premises/equipment qualification and monitoring, raw materials management, starting materials, technical manufacturing processes, quality controls, and the release, thawing and infusion were substantially reorganized. Numerous studies have been carried out to validate cultures and demonstrate the short-term stability of fresh or thawed products, as well their stability during long-term storage. Detailed results of media simulation tests, validation runs and early MSC batches are presented. We also report the validation of a new variant of the process aiming to prepare fresh MSCs for the treatment of specific lesions of Crohn’s disease by local injection. In conclusion, we have successfully ensured the adaptation of our clinical-grade MSC production process to the GMP requirements. The GMP manufacturing of MSC products is feasible in the academic setting for a limited number of batches with a significant cost increase, but moving to large-scale production necessary for phase III trials would require the involvement of industrial partners

Low T-cell chimerism is not followed by graft rejection after nonmyeloablative stem cell transplantation (NMSCT) with CD34-selected PBSC.

peer reviewedWe investigate the feasibility of CD34-selected peripheral blood stem cell (PBSC) transplantation followed by pre-emptive CD8-depleted donor lymphocyte infusions (DLI) after a minimal conditioning regimen. Six patients with advanced hematological malignancies ineligible for a conventional myeloablative transplant (n=5) or metastatic renal cell carcinoma (n=1), and with an HLA-identical (n=4) or alternative (n=2) donor were included. The nonmyeloablative conditioning regimen consisted in 2 Gy TBI alone (n=4), 2 Gy TBI and fludarabine (RCC patient, n=1) or cyclophosphamide and fludarabine (patient who had previously received 12 Gy TBI, n=1). Post transplant immunosuppression was carried out with cyclosporin (CyA) and mycophenolate mofetil (MMF). Initial engraftment was achieved in all patients. One out of six patients (17%) experienced grade > or =2 acute GVHD only after abrupt cyclosporin discontinuation and alpha interferon therapy for life-threatening tumor progression. T-cell chimerism was 23% (19-30) on day 28, 32% (10-35) on day 100, 78% (49-95) on day 180 and 99.5% (99-100) on day 365. Three out of four patients who had measurable disease before the transplant experienced a complete response. We conclude that CD34-selected NMSCT followed by CD8-depleted DLI is feasible and preserves engraftment and apparently also the graft-versus-leukemia (GVL) effect. Further studies are needed to confirm this encouraging preliminary report

Administration of Third-Party Mesenchymal Stromal Cells at the Time of Kidney Transplantation: Interim Safety Analysis at One-Year Follow-Up

Mesenchymal stromal cells (MSC)-based therapy has been proposed in kidney transplantation (KTx). We report on the 1-year follow-up of an open-label phase I trial using MSC in KTx. On postoperative day 3, third-party MSC (~2.0x106/kg) were administered to 7 non-immunized first-transplant recip- ients from deceased donors, under standard immunosuppression (Basiliximab, Tacrolimus, MMF and steroids). No HLA matching was required for MSC donors. Seven comparable KTx recipients were included as controls. Informed consent was obtained. No side-effect was noted at the time of MSC injection. Still, 1 patient with a history of ischemic heart disease had a NSTEMI ~3h after MSC infusion. Ten months after KTx, 1 MSC patient had type B aortic dissection and STEMI. Four MSC patients had at least 1 opportunistic infection, whereas 3 controls had polyoma-BK viremia. At day 14, eGFR in MSC and control groups was 47.1 ± 6.8 and 39.7 ± 5.9 ml/min, respectively (p, 0.05). At 1 year, eGFR in MSC and control groups was 46.5 ± 18.6 and 54.2 ± 16.3 ml/min, respectively (p, 0.42). Per-cause biopsies evidenced 1 bor- derline and 1 acute rejections in MSC group, whereas no AR was biopsy-proven in controls. Three patients developed anti-HLA antibodies against MSC (n=1) or shared kidney/MSC (n=2) mismatches.MSC infusion was safe in all patients except one. Incidence of opportunist infections was similar in both groups. No difference in eGFR was found at 1-year post KTx. Putative immunization against MSC was observed in 3 patients

2002; 01

DLI is feasible with rapid engraftment and minimizes the risk of severe GVHD. Large prospective trials are required to prove that it preserves the GVL effect fully. ©2002, Ferrata Storti Foundation Key words: allogeneic transplantation, GVHD, GVL, CD34 selection, donor lymphocyte infusions. plication of DLI is GVHD, which is often associated with response to DLI. © F e r r a t a S t o r t i F o u n d a t i o n Design and Methods Patients and donors Twenty-four patients with hematologic malignancies, 18 males and 6 females, aged 14 to 56 years (median 46 years) were included. Their clinical characteristics are summarized in © F e r r a t a S t o r t i F o u n d a t i o n obtained from patients and donors and our institution's Ethical Committee approved the protocol. Clinical management The conditioning regimen depended on the clinical diagnosis. The following regimens were used: 1) Ara-C (12 g/m 2 ), cyclophosphamide (120 mg/m 2 ) and single dose total body irradiation-TBI (8 Gy) (AML and MDS patients); 2) Ara-C (18 g/m 2 ), melphalan (140 mg/m 2 ) and fractionated TBI (12 Gy) (ALL patients); 3) busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (CML and 2 nd transplant patients); 4) cyclophosphamide (120 mg/kg) and fractionated TBI (12 Gy) (NHL and CLL patients). All patients were treated with 5 µg/kg/d lenograstim (Granocyte®) from day +1 until the granulocyte count was > 10 9 /L for three consecutive days or > 10 10 /L for one day. Six early patients (4 not HLA-identical to their donor and 2 AML in CR1) received a short course of methotrexate in addition to cyclosporine (CyA). Because of the low incidence of acute GVHD observed in the first 9 patients, GVHD prophylaxis was carried out with CyA alone for patients 10 to 24. The diagnosis and grading of acute and chronic GVHD was established as previously reported. Stem cell mobilization, collection and selection Donors received human granulocyte colonystimulating factor (G-CSF) (Granocyte®, kindly provided by Rhône-Poulenc-Rorer, Brussels, Belgium) at a dose of 10-15 µg/kg from day -5 through day -1 before transplant. Collection of PBSC was carried out on days -1 and 0, using a continuous flow blood cell separator (CS3000+, Baxter-Fenwall Laboratories, Deerfield, IL, or Cobe Spectra, Lakewood, CO, USA). The volume of blood processed was 12-16 liters. The PBSC from the first day of harvest were stored overnight in the patient's own plasma. Immediately after the second harvest, PBSC from the first and the second days of harvest were pooled. CD34 + cell selection was carried out using the Isolex 300i® magnetic cell separator (Nexell International, Wemmel, Belgium), according to the manufacturer's recommendations. Donor lymphocyte infusions Around day 60 post-transplantation, donors underwent 12-16 liter leukophereses on 2 consecutive days to collect lymphocytes. The collection from the first day of harvest was stored overnight in the patient's own plasma and pooled with the second harvest before processing by CD8 + selection using the Nexell Isolex 300i®. The CD8-negative fraction was recovered and divided into 3 aliquots containing 2×10 6 , 1×10 7 and 5×10 7 CD3 + cells/kg recipient for patients from #1 to #13. The 2 nd and 3 rd aliquots were cryopreserved in 10% DMSO in a controlled-rate freezer. After an interim analysis showed little acute or chronic GVHD associated with DLI, the schedule of DLI was changed to 2 aliquots of 1×10 7 and 5×10 7 CD3 + cells/kg recipient for patients 14 to 21. Aliquot 1 was injected fresh immediately after the CD8 depletion procedure (around day 60). Around day 100 (and 140 for patients 1 to 13), aliquot 2 (and 3) were thawed and infused into the patient. CD8-depleted DLI were not to be infused in case of an antecedent grade III or IV acute GVHD, or an antecedent extensive chronic GVHD, or active GVHD at the time of the scheduled infusion. Laboratory analyses Aliquots of the pooled PBSC as well as the CD34 + selected fraction were incubated with phycoerythrin-conjugated anti-CD34 monoclonal antibody (HPCA2; Becton-Dickinson, Palo-Alto, CA, USA) for 20 minutes at 20°C, washed and fixed. A total of 1×10 5 cells was analyzed using a FACS-scan analyzer (Becton-Dickinson). The percentage of CD34 + cells was defined with dot plot analysis using the whole nucleated cell population. The percentage of positive cells in the isotype control was subtracted from the CD34 + percentage to give the final percentage of CD34 + cells. Data acquisition was performed with the Cellquest software (BectonDickinson). Donor lymphocytes (before and after CD8 depletion) were similarly examined using double labeling with FITC-and PE-conjugated antibodies after treatment with a lysing solution. Complete blood counts were determined using a Technicon H2 cell counter (Bayer Diagnostics, Diegem, Belgium). Percentages of reticulocytes were obtained by an automated cytofluorometric method using the thiazole orange analog DEQTC. Statistical analyses Student's t-tests were used to compare cell subsets before and after CD8 depletion. The probability of GVHD, relapse, and survival as well as the speed of engraftment were studied by life-table analyses and Wilcoxon rank tests were used for comparisons between groups. Statistical analyses were carried out with Graphpad Prism (Graphpad Software, San Diego, CA, USA). Results Collection of PBSC, CD34 selection and engraftment kinetics PBSC were collected by leukophereses on two consecutive days, except in one 70-year old donor (patient #6) who had to undergo 4 consecutive leukophereses (and two CD34 selection procedures) because of poor yields. Most donors experienced bone pain and/or cephalalgia that were easily controlled with paracetamol, but no other complication was noted. A median of 10.15 (5.59 to 21.02) ×10 6 CD34 + cells/kg and 375 (127 to 656)×10 6 CD3 + cells/kg were collected CD8-depleted DLI after CD34-selected allo-PBSCT © F e r r a t a S t o r t i F o u n d a t i o n Collection of donor lymphocytes, CD8 depletion and CD8-depleted DLI Donor lymphocytes were collected on day 60 from all but 5 donors whose recipients died before or experienced serious complications Clinical data (Figure 1 and Table 4) Five patients died before receiving DLI. Patients #6 (second transplant) and #4 developed many early serious complications and died on day 81 of influenza pneumonia and on day 99 of a polymicrobial infection, respectively. Patient #19 developed severe renal and respiratory failure and died of infection at day 195. Patient #16 died of CLL and veno-occlusive disease of the liver on day 44 and patient #17 died of leukemia on day 38. The other 19 patients were in CR on day 60 and received the first DLI. Two ALL patients (patients #7 and 10) relapsed between days 60 and 100 and received the pooled 2 nd and 3 rd aliquots. Patient #7 achieved a CR with chemotherapy and DLI but relapsed later and died on day 313. Patient #10 died of leukemia on day 100. In addition, patient #12 died suddenly at home of unknown reason while enjoying continuous CR and presenting no complication other than depression. The other 16 received the scheduled 2 nd (and the 3 rd for patients #1, 2, 3, 5, 8, 9, 10, 13) DLI on days 100 (and 140). Fourteen of the 16 patients remain in CR 126 to 1344 (median 400) days post-transplantation. Patient #15 developed a central nervous system relapse on day 135, was reinduced into remission with chemotherapy and radiotherapy but relapsed again and died on day 309. Patient #3 experienced a biopsyproven massive relapse in the thymus on day 315 that completely regressed after cyclosporine discontinuation, but later relapsed again and she died on day 950. Finally, 3 patients died in CR after day 100: patient #13 died of pulmonary aspergillosis on day 351, patient #18 died suddenly at home of unknown reason on day 288 and patient #21 of acute grade IV GVHD on day 150. The remaining 11 patients were alive and disease-free 126 to 1344 (median 550) days after transplantation. Bone marrow chimerism Evaluation of bone marrow chimerism was performed in 6/7 patients with a donor of the opposite sex (n=7) by FISH with X and Y probes ( Acute and chronic GVHD (Figure 3) Acute GVHD occurred in 15 patients. It was of grade I in 9 patients, of grade II in 5 patients (3 with 1 HLA mismatch) and of grade IV in 1 patient (HLA mismatch). Thus, grade II-IV acute GVHD occurred more frequently in HLA-mismatched (4/8 or 50%) than HLA-matched (2/16 or 13%) transplants (p<0.05). Before DLI, the 60-day actuarial incidence of grade II-IV GVHD was 17% (4 patients) but 2 additional patients developed grade II and IV GVHD after DLI to produce a 150-day (after DLI) incidence of 28%. For HLA-identical sibling transplants, the 60-and 150-day actuarial incidences of grade II-IV acute GVHD were 0 and 13%, respectively ( CMV reactivation Eleven of the 24 patients experienced CMV reactivation (PCR positivity) before day 60 that was successfully reversed by ganciclovir treatment and none of them presented a clinical CMV infection. Relapse and survival (Figure 4) There was no relapse in the 12 standard risk patients but 6 of the high-risk patients relapsed (p<0.005) ( Discussion In agreement with previous studies, 17,19,21 CD34-selection resulted in our study in a 3.3 log elimination of T-cells while preserving hematologic reconstitution. Engraftment of neutrophils and platelets was prompt and significantly faster in patients who did not receive methotrexate. However, within the range studied, the number of © F e r r a t a S t o r t i F o u n d a t i o n CD34 + cells did not influence the speed of engraftment significantly. Prompt engraftment occurred with CD34 + cell doses as low as 1.46×10 6 /kg. Therefore, CD34 -selection preserved the engraftment capability of allogeneic PBSC. As previously suggested by others, 17,22 our results evidenced that CD34 selection reduces the risk of acute GVHD. The actuarial 60-day (before DLI) probability of grade II-IV acute GVHD was 17% in our study. This rate compares very favorably with results from studies of HLA-identical siblings receiving unmanipulated PBSC or BM. Moreover, our results also compared favorably with those of previous studies of pre-emptive DLI after T-cell-depleted BMT or PBSC transplantation. A few other studies have investigated the feasibility of adding T-cells back, also to heterogeneous groups of patients, a few weeks to a few months after T-cell-depleted (TCD) transplantation. Barrett gave either 2×10 6 /kg on day 30 and 5×10 7 /kg on day 45 or 1×10 7 /kg on day 30 to HLA-identical siblings after TCD BMT. Contributions and Acknowledgments YB designed the study and wrote the paper. FB analyzed the data and wrote the paper. JS © F e r r a t a S t o r t i F o u n d a t i o n PEER REVIEW OUTCOMES What is already known on this topic Transplantation of CD34 + selected cells from peripheral blood of allogeneic donors associates with a lower risk of acute and chronic graft-versus-host disease than unmanipulated transplants. Relapse risk may be increased in this setting, as a consequence of T-cell depletion, which leads to a decreased graft-versusleukemia effect. What this study adds CD8-depleted lymphocyte infusions starting on day 60 may be safely administered without triggering acute or chronic graft-versus-host disease. The relapse rate after this approach was similar to that observed in a control group of non-T-cell depleted transplants. Manuscript processing Potential implications for clinical practice This report demonstrates the feasibility of allogeneic CD34 + selected stem cell transplantation followed by the infusion of engineered cell-depleted fractions. Jordi Sierra Gil, Deputy Editor © F e r r a t a S t o r t i F o u n d a t i o

Bone Marrow-Derived Mesenchymal Stromal Cell Therapy in Severe COVID-19: Preliminary Results of a Phase I/II Clinical Trial

peer reviewedBackground: Treatment of acute respiratory distress syndrome (ARDS) associated with COronaVIrus Disease-2019 (COVID-19) currently relies on dexamethasone and supportive mechanical ventilation, and remains associated with high mortality. Given their ability to limit inflammation, induce immune cells into a regulatory phenotype and stimulate tissue repair, mesenchymal stromal cells (MSCs) represent a promising therapy for severe and critical COVID-19 disease, which is associated with an uncontrolled immune-mediated inflammatory response. Methods: In this phase I-II trial, we aimed to evaluate the safety and efficacy of 3 intravenous infusions of bone marrow (BM)-derived MSCs at 3-day intervals in patients with severe COVID-19. All patients also received dexamethasone and standard supportive therapy. Between June 2020 and September 2021, 8 intensive care unit patients requiring supplemental oxygen (high-flow nasal oxygen in 7 patients, invasive mechanical ventilation in 1 patient) were treated with BM-MSCs. We retrospectively compared the outcomes of these MSC-treated patients with those of 24 matched control patients. Groups were compared by paired statistical tests. Results: MSC infusions were well tolerated, and no adverse effect related to MSC infusions were reported (one patient had an ischemic stroke related to aortic endocarditis). Overall, 3 patients required invasive mechanical ventilation, including one who required extracorporeal membrane oxygenation, but all patients ultimately had a favorable outcome. Survival was significantly higher in the MSC group, both at 28 and 60 days (100% vs 79.2%, p = 0.025 and 100% vs 70.8%, p = 0.0082, respectively), while no significant difference was observed in the need for mechanical ventilation nor in the number of invasive ventilation-free days, high flow nasal oxygenation-free days, oxygen support-free days and ICU-free days. MSC-treated patients also had a significantly lower day-7 D-dimer value compared to control patients (median 821.0 µg/L [IQR 362.0-1305.0] vs 3553 µg/L [IQR 1155.0-6433.5], p = 0.0085). Conclusions: BM-MSC therapy is safe and shows very promising efficacy in severe COVID-19, with a higher survival in our MSC cohort compared to matched control patients. These observations need to be confirmed in a randomized controlled trial designed to demonstrate the efficacy of BM-MSCs in COVID-19 ARDS. Clinical Trial Registration (www.ClinicalTrials.gov), identifier NCT0444545

Effect of HLA-matching recipients to donor non-inherited maternal antigens on outcomes after mismatched umbilical cord blood transplantation for hematologic malignancy

peer reviewe