Hematopoietic stem cell transplantation (HSCT) from haploidentical donors is a viable option for patients lacking HLA-matched donors. Here we report the results of a prospective multicenter phase I/II trial of transplantation of TCRαβ and CD19-depleted peripheral blood stem cells from haploidentical family donors after a reduced-intensity conditioning with fludarabine, thiotepa, and melphalan. Thirty pediatric and 30 adult patients with acute leukemia ( n = 43), myelodysplastic or myeloproliferative syndrome ( n = 6), multiple myeloma ( n = 1), solid tumors ( n = 6), and non-malignant disorders ( n = 4) were enrolled. TCR αβ/CD19-depleted grafts prepared decentrally at six manufacturing sites contained a median of 12.1 × 10 6 CD34 + cells/kg and 14.2 × 10 3 TCRαβ + T-cells/kg. None of the patients developed grade lll/IV acute graft-versus-host disease (GVHD) and only six patients (10%) had grade II acute GVHD. With a median follow-up of 733 days 36/60 patients are alive. The cumulative incidence of non-relapse mortality at day 100, 1 and 2 years after HSCT was 5%, 15%, and 17% for all patients, respectively. Estimated probabilities of overall and disease-free survival at 2 years were 63% and 50%, respectively. Based on these promising results in a high-risk patient cohort, haploidentical HSCT using TCRαβ/CD19-depleted grafts represents a viable treatment option.

Background & Aim Major complications of allogeneic stem cell transplantation are GvHD and infections. T cell depletion (TCD) can effectively prevent GVHD, however it may prolong immune reconstitution and increase the risk of infections. Depletion of TCRab+ T cells combined with the transfer of CD45RA− donor memory T cells can ameliorate the infection risk and simultaneously decrease the risk of GVHD. To generate robust, fully automated and scalable cell manufacturing procedures for high quality personalized grafts, we report the development of a single automated process on the CliniMACS Prodigy®. Methods, Results & Conclusion The CliniMACS Prodigy currently employs two separate applications: The combined depletion of TCRab+/CD19+ cells and the depletion of CD45RA+ cells. The novel integrated CliniMACS Prodigy LP-TCRab-19-45RA depletion process merges these two strategies in one process. Depending on the desired cell composition of the graft, the user can choose to perform either a sequential depletion of CD45RA+ and TCRab+ cells with or without simultaneous depletion of CD19+ cells, a depletion of TCRab+ cells with or without simultaneous CD19+ cells alone or a CD45RA+ cell depletion only. Every application case generates separate memory T cell and stem cell products that can be combined to a final cell product, according to the patient need (personalized graft). With this process, we can effectively deplete 2.4 × 109 CD45RA+ cells from up to 6 × 109 total WBC, and 48 × 109 total TCRab+ cells plus 15 × 109 total CD19+ cells from up to 120 × 109 total WBC. The depletion performance was tested by inhouse verification and external validation runs (n=10) and yielded a mean depletion of 5.8 log (range 5.1 – 6.5) for CD45RA+ cells and 4.2 log (range 3.5 – 5.2) for TCRab+ cells with a stem cell viability and recovery of over 85%. Preliminary results indicate that combined depletion of CD19+ cells resulted in comparable values for depletion, yield, recovery and WBC viability of the target products. For quality control an automated Express Mode flow cytometric analysis software using the MACSQuant® Analyzer was developed enabling fast, accurate and reproducible results. In summary, the novel automated CliniMACS Prodigy LP-TCRαβ-19-45RA depletion process is capable to deplete CD45RA+, TCRab+ and CD19+ cells efficiently from apheresis products with a mean recovery of CD34+ cells of 85%. The performance verification and submission to an European notified body for CE mark are the important next steps. Major complications of allogeneic stem cell transplantation are GvHD and infections. T cell depletion (TCD) can effectively prevent GVHD, however it may prolong immune reconstitution and increase the risk of infections. Depletion of TCRab+ T cells combined with the transfer of CD45RA− donor memory T cells can ameliorate the infection risk and simultaneously decrease the risk of GVHD. To generate robust, fully automated and scalable cell manufacturing procedures for high quality personalized grafts, we report the development of a single automated process on the CliniMACS Prodigy®. The CliniMACS Prodigy currently employs two separate applications: The combined depletion of TCRab+/CD19+ cells and the depletion of CD45RA+ cells. The novel integrated CliniMACS Prodigy LP-TCRab-19-45RA depletion process merges these two strategies in one process. Depending on the desired cell composition of the graft, the user can choose to perform either a sequential depletion of CD45RA+ and TCRab+ cells with or without simultaneous depletion of CD19+ cells, a depletion of TCRab+ cells with or without simultaneous CD19+ cells alone or a CD45RA+ cell depletion only. Every application case generates separate memory T cell and stem cell products that can be combined to a final cell product, according to the patient need (personalized graft). With this process, we can effectively deplete 2.4 × 109 CD45RA+ cells from up to 6 × 109 total WBC, and 48 × 109 total TCRab+ cells plus 15 × 109 total CD19+ cells from up to 120 × 109 total WBC. The depletion performance was tested by inhouse verification and external validation runs (n=10) and yielded a mean depletion of 5.8 log (range 5.1 – 6.5) for CD45RA+ cells and 4.2 log (range 3.5 – 5.2) for TCRab+ cells with a stem cell viability and recovery of over 85%. Preliminary results indicate that combined depletion of CD19+ cells resulted in comparable values for depletion, yield, recovery and WBC viability of the target products. For quality control an automated Express Mode flow cytometric analysis software using the MACSQuant® Analyzer was developed enabling fast, accurate and reproducible results.

Abstract Abstract: Here we report for the first time on long term follow-up data of a cohort of 60 patients who received TCRαβ and CD19 depleted peripheral blood stem cell grafts from haploidentical family donors within a prospective, multi-center, single-arm, phase I/II clinical study (EudraCT No.: 2011-005562-38). As planned, 30 pediatric and 30 adult patients were enrolled in this trial: All patients received a reduced-intensity conditioning regimen consisting of fludarabine (160 mg/m2), thiotepa (10 mg/kg), melphalan (140 mg/m2) and either antithymocyte globulin (Grafalon, 15 or 30 mg/kg, N=53) or 7 Gy total nodal irradiation (N=7). MMF (40 mg/kg/day) was administered as single-agent GVHD prophylaxis until Day 30. Results: Sixty patients with a median age of 18.5 years (range 1-63) were treated. Twenty-five patients had AML, 17 ALL, 6 MDS/MPS and 1 each had multiple myeloma and acute undifferentiated leukemia. Six patients had solid tumors (soft tissue sarcomas and neuroblastomas) and 4 non-malignant disorders (SCID, Wiskott-Aldrich syndrome, lysosomal storage disorder and sickle cell anemia). Of the 56 patients with malignant disease, 33 were transplanted in complete (CR), 11 in partial (PR) and 12 in non-remission (NR). Twenty of the 56 patients with malignant diseases received a 2nd or 3rd transplantation within this protocol. In total, 88 depletion procedures were performed with the CliniMACS plus System (Miltenyi Biotec, Germany) at 7 GMP laboratories and resulted in a median T and B cell log depletion of 4.7 (range 3.6-5.3) and 3.4 (range: 2.3-4.5), respectively. The median number of infused CD34+ cells and TCRαβ T cells was 12.4 × 106 /kg BW (range 4.0 - 54.9) and 1.4× 104 /kg BW (range 0.06-6.4), respectively. Engraftment was rapid with a median of 13 (range 9-41) and 15 (11-38) days to reach ANC >500 cell/µl and PLT > 20,000 cells/µl. Nine patients rejected the graft. Eight of them were successfully re-transplanted and 1 patient died. One patient received stem cell boosts from the original donor due to poor graft function. On day 100, peripheral T cell chimerism was completely donor-type in 44 of 47 evaluable patients, and mixed in 3. None of the patients developed grade III/IV aGVHD and only 6 patients (10%) had grade II aGVHD. Of 47 evaluable patients 4 had severe cGVHD (9%), and 6 (13%) and 5 (11%) had moderate and mild cGVHD, respectively. CMV reactivation was seen in 25 (42%) mainly adult patients, and only 1 (2%) patient developed disease. Twenty-one (35%) patients had ADV reactivation and 7 children and 1 adult (13%) developed disease. Only 1 case of EBV disease (encephalitis) occurred (2%). A median of 221 (range 8-1230) CD3+ cells/µl was reached on day 100. The median numbers of CD3/CD4+ and CD3/CD8+ cells at 1 year post transplant were 316 (range 1-1173) and 308 (range 0-2203) /µl. As of July 15 2018, 57 patients have completed the 2 year follow-up, died or discontinued the study resulting in a median follow-up of 706 days (range 18-800). 37 patients (62%) are alive and 23 (38%) died. Relapse was the major cause of death (N=12) followed by ADV infection (N=3), ARDS (N=3) and 1 case each of cardiac arrest, multi organ failure, sepsis due to graft failure and demyelinating neuropathy. Cause of death was not reported in 1 patient. Eight of the 20 patients who received the 2nd or 3rd transplantation are alive; 1 discontinued the study prematurely. Of the 23 patients transplanted in PR or NR, 13 are alive. The Kaplan-Meier estimated probabilities of overall survival, disease-free survival (DFS) were 62% and 53%, respectively. Cumulative incidences of relapse and NRM at 2 years were 34% and 20%, respectively. For those patients with leukemia receiving a first SCT in CR, the overall survival, DFS and relapse rate were 75%, 64% and 20%, respectively. Conclusion: The transplantation of TCRαβ and CD19 depleted haploidentical hematopoietic stem cell grafts was safe and feasible. Decentralized production using the CliniMACS System was feasible and reliably resulted in grafts containing sufficient numbers of stem cells with only minimal numbers of co-infused TCRαβ T cells. None of the patients developed grade III-IV aGVHD and incidence of cGVHD was acceptable. Given the heterogeneous patient cohort with respect to age, disease, remission status and number of previous transplants, the outcome of patients after 2 years follow-up is promising. Disclosures Lang: Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Meisel:Amgen: Consultancy. Mielke:KIADIS Pharma: Speakers Bureau; Miltenyi Biotec: Speakers Bureau; DGHO: Speakers Bureau; EHA: Speakers Bureau; Celgene: Speakers Bureau. Niederwieser:Novartis: Research Funding; Miltenyi: Speakers Bureau. Bader:Neovii: Research Funding; Medac: Patents & Royalties, Research Funding; Riemser: Research Funding; Cellgene: Consultancy; Novartis: Consultancy, Speakers Bureau. Kuball:Gadeta (www.gadeta.nl): Consultancy, Equity Ownership, Patents & Royalties: on gd T cells and receptors and isolation strategies, Research Funding; Miltenyi Biotec: Research Funding; Novartis: Research Funding. Bonig:Miltenyi Biotec GmbH: Honoraria, Research Funding. Karitzky:Miltenyi Biotec GmbH: Employment. Holtkamp:Miltenyi Biotec GmbH: Employment. Malchow:Miltenyi Biotec GmbH: Employment. Siewert:Miltenyi Biotec GmbH: Employment. Biedermann:Miltenyi Biotec GmbH: Employment. Bethge:Neovii GmbH: Honoraria, Research Funding; Miltenyi Biotec GmbH: Consultancy, Honoraria, Research Funding.

Stem cells have the potential to revolutionize tissue regeneration and engineering. Both general types of stem cells, those with pluripotent differentiation potential as well as those with multipotent differentiation potential, are of equal interest. They are important tools to further understanding of general cellular processes, to refine industrial applications for drug target discovery and predictive toxicology, and to gain more insights into their potential for tissue regeneration. This chapter provides an overview of existing sorting technologies and protocols, outlines the phenotypic characteristics of a number of different stem cells, and summarizes their potential clinical applications.