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Miltenyi Biotec distribution:
May 28–29, 2020
Meet us at the first virtual meeting of the ISCT
The current developments regarding the COVID-19 pandemic force all of us to rethink our way of working and living. Especially when it comes to bringing people together, networking, and exchanging ideas on a professional level, creative solutions are needed. Therefore, we are delighted to take part in the first virtual meeting of the ISCT. Join us in looking forward to a broad scientific program and get connected with experts from all over the world. Don’t miss out on our symposium discussing “Advanced solutions for cutting-edge cell therapies” which will be accessible to ISCT attendees for 12 months.
CliniMACS Prodigy® Adherent Cell Culture System
May 28, 2020 | May 29, 2020
Join the virtual demo and learn how to automate adherent stem cell manufacturing in a closed system.
Advanced solutions for cutting-edge cell therapies
Thursday, May 28, 11:15 a.m.–12:45 p.m. PDT (Seattle)/08:15 p.m.–09:45 p.m. CEST
Sort-purification of human CD34+CD90+ cells reduces target cell population and improves lentiviral transduction for gene therapy
Hematopoietic stem cell (HSC) gene therapy is a promising treatment option for various hematological diseases and disorders. Most currently available approaches target CD34+ cell–enriched fractions, a heterogeneous mix of mostly committed progenitor cells and only very few true HSCs with long-term multilineage engraftment potential. Consequently, gene therapy approaches are limited in their HSC targeting efficiency and very expensive due to the large quantities of modifying reagents needed. In addition, exposure of non-target cells to lentiviral vectors or nucleases can increase the risk of unwanted side-effects in these cell populations.
We aimed to develop a clinical protocol to reliably purify and efficiently gene-modify human HSC-enriched CD90+ cell fractions, which ultimately would allow us to reduce costs without compromising in vivo engraftment.
Large-scale enrichment of CD34+ cells from GCSF-mobilized leukapheresis products was initially performed on Miltenyi Biotec’s CliniMACS Prodigy®. Yield, purity, quality, and feasibility of CD90+ cell sorting was then tested on the jet-in-air sorter FX500 from Sony and the cartridge-based closed-system sorter MACSQuant® Tyto® from Miltenyi Biotec. Transduction was performed using a clinical-grade, GFP-encoding lentivirus. Engraftment was tested using the NSG mouse xenograft model.
Purity and yield after flow cytometric sorting of CD90+ cells were similar with either the FX500 or MACSQuant® Tyto®. Both approaches reliably reduced the overall target cell count by 10- to 15-fold without impacting the cells’ viability and in vitro colony-forming cell potential. Transduction efficiency of sorted CD90+ cells was significantly improved compared to bulk CD34+ and especially the CD34+CD90+ subset. All cell fractions demonstrated robust mouse xenograft potential. Significantly higher levels of GFP expression in peripheral blood, bone marrow, spleen, and thymus were observed after transplantation of gene-modified CD90+ compared to bulk CD34+ cells in NSG mice.
Advanced cell therapy based on the chimeric receptor NKG2D: the NK-T cell approach
NKG2D is a C‐type lectin‐like transmembrane activating receptor present on the surface of natural killer (NK) cells, NKT cells, CD8+TCRγδ+ T cells, and certain subsets of CD4+ T cells. Biogenesis of NKG2D ligands (NKG2DL) is stimulated in cells under stress conditions such as viral infection, cellular senescence, and tumorigenesis. NKG2DL are expressed on various tumor types including different pediatric solid and hematological malignancies, thus providing suitable targets for cancer therapy. NKG2D activation on NK cells results in cytokine secretion and exocytosis of cytotoxic granules. NKG2D is particularly relevant for cancer immunosurveillance: Interaction between NKG2DL and NKG2D receptor is essential for NK cell–mediated elimination of osteosarcoma tumor‐initiating cells. However, tumor cells can develop various immune escape strategies. Nevertheless, the use of NKG2D‐CAR on memory CD45RA– T cells may overcome these limitations. We have shown that NKG2D‐CAR–expressing CD45RA– T cells were cytotoxic against three osteosarcoma cell lines and 8/10 leukemia cell lines with specific lysis of over 50%. Myeloid and T‐ALL cell lines were more susceptible (specific lysis ranging from 50–78%) than B‐ALL cell lines (19–52%). NKG2D‐CAR+ memory CD45RA– T cells also had considerable antitumor activity in a mouse model of human osteosarcoma, whereas non-transduced T cells were ineffective. We have developed a protocol to expand clinical-grade NKG2D‐CAR–expressing memory CD45RA– T cells in a fully automated closed system, CliniMACS Prodigy®. This expansion protocol allowed us to obtain up to 2076±697 million cells with 77.8±20% NKG2D‐CAR expression and 76±10% viability. Harvested CAR T cells showed specific lysis of Jurkat cells (90±14%) and 531MII osteosarcoma cell line (31±16%). Vector copy number was ≤5 in all validations. CGH and karyotype showed no genetic alterations. Free viral particles were undetectable in the supernatants. No overexpression of MYC/TERT was found except for one validation. Endotoxins were ≤0.25 EU/mL. Automated manufacturing of clinical‐grade NKG2D‐CAR–expressing memory CD45RA– T cells using the CliniMACS Prodigy® is feasible and reproducible. We plan to explore different clinical trials on pediatric diseases.
Manufacturing of induced pluripotent stem cells and immune-derived cells
The breakthrough discovery of induced pluripotent stem cells (iPSCs) is currently profoundly modifying the landscape of cell therapy and allows us to open novel perspectives for the generation of advanced therapy medicinal products (ATMP) potentially applicable in all fields of medicine. In terms of a large-scale therapeutic landscape, implementation of iPSC-derived allogeneic therapies will require suitable immune-HLA-matched iPSC lines from healthy universal donors and/or the derivation of hypoimmunogenic iPSC lines. To anticipate future demands in effective allogeneic therapies, the availability of accessible cost-effective and safe iPSC lines is a major requirement, to provide off-the-shelf unlimited numbers of therapeutic products from a single iPSC master cell bank. We have previously developed several research-grade master banks of human iPSCs using an in vitro expansion workflow. However, several hurdles remain for industrial, cGMP-grade, large-scale production and banking of these cells.
We report a procedure using the integrated GMP-compliant cell processing platform CliniMACS Prodigy® providing automated cell feeding and harvesting in a closed system. The iPSC clone used on the CliniMACS Prodigy Platform was previously derived from a healthy donor using the CytoTune™- iPS 2.0 Sendai Reprogramming Kit, manufactured according to GMP principles, on StemMACS™ iPS-Brew XF, human medium and human recombinant laminin matrix. Over 14 in vitro cell passages, this cell line was replated and expanded on the CliniMACS Prodigy Platform within 2 weeks. At the end of the process, 1.4 billion iPSCs were collected with a high genetic stability as evaluated by karyotyping and CNV/NGS analysis before and after scalable expansion. Expanded iPSCs maintained their pluripotency markers and an efficient differentiation towards endodermal, mesodermal, and ectodermal trilineage layers. Overall, this process provides a safe and standardized scalable manufacturing platform for GMP-iPSC master cell banks. The feasibility of this procedure opens novel perspectives in large-scale production of mesenchymal stem cells and immunocompetent cells for our therapeutic program.
The statements and other contributions presented at the meeting and the views expressed therein are those of the attending experts and do not necessarily represent the policy or opinion of Miltenyi Biotec or any institution the respective expert may be associated with. Neither Miltenyi Biotec nor any of its directors or employees give any representation or warranty as to the reliability, accuracy, or completeness of the content thereof. Participants have an implied responsibility to use the newly acquired information to enhance patient outcomes and their own professional development. The information presented in this activity is not meant to serve as a guideline for patient management. Any procedures, medications, or other courses of diagnosis or treatment discussed or suggested in this activity should not be used by clinicians without evaluation of their patient’s conditions and possible contraindications on dangers in use, review of any applicable manufacturer’s product information, and comparison with recommendations of other authorities. This notice must accompany any further distribution of this document.
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