Anita Schmitt

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMSnHuman mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process.nnnMETHODSnThis report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production.nnnRESULTSnThe strategy for quality control testing depends on the products cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs.nnnCONCLUSIONSnThis position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Biosimilar G-CSF based mobilization of peripheral blood hematopoietic stem cells for autologous and allogeneic stem cell transplantation

The use of granulocyte colony stimulating factor (G-CSF) biosimilars for peripheral blood hematopoietic stem cell (PBSC) mobilization has stimulated an ongoing debate regarding their efficacy and safety. However, the use of biosimilar G-CSF was approved by the European Medicines Agency (EMA) for all the registered indications of the originator G-CSF (Neupogen®) including mobilization of stem cells. Here, we performed a comprehensive review of published reports on the use of biosimilar G-CSF covering patients with hematological malignancies as well as healthy donors that underwent stem cell mobilization at multiple centers using site-specific non-randomized regimens with a biosimilar G-CSF in the autologous and allogeneic setting. A total of 904 patients mostly with hematological malignancies as well as healthy donors underwent successful autologous or allogeneic stem cell mobilization, respectively, using a biosimilar G-CSF (520 with Ratiograstim®/Tevagrastim, 384 with Zarzio®). The indication for stem cell mobilization in hematology patients included 326 patients with multiple myeloma, 273 with Non-Hodgkins lymphoma (NHL), 79 with Hodgkins lymphoma (HL), and other disease. 156 sibling or volunteer unrelated donors were mobilized using biosimilar G-CSF. Mobilization resulted in good mobilization of CD34+ stem cells with side effects similar to originator G-CSF. Post transplantation engraftment did not significantly differ from results previously documented with the originator G-CSF. The side effects experienced by the patients or donors mobilized by biosimilar G-CSF were minimal and were comparable to those of originator G-CSF. In summary, the efficacy of biosimilar G-CSFs in terms of PBSC yield as well as their toxicity profile are equivalent to historical data with the reference G-CSF.

Rescue stem cell mobilization with plerixafor economizes leukapheresis in patients with multiple myeloma

While extensive data demonstrated that plerixafor improves stem cell harvest in difficult‐to‐mobilize patients, economic concerns limit a broader application. We retrospectively assessed the effect of an early plerixafor rescue regimen for mobilization in patients with multiple myeloma. Patients were intended for high‐dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ABSCT) and therefore received cyclophosphamide‐based mobilization chemotherapy and consecutive stimulation with granulocyte colony‐stimulating factor (G‐CSF). Fifteen patients with poor stem cell harvest in the first leukapheresis session received plerixafor. Data were compared with a matched historic control group of 45 patients who also had a poor stem cell yield in the first apheresis session, but continued mobilization with G‐CSF alone. Patients in the plerixafor group collected significantly more CD34+ cells in total (median 4.9 vs. 3.7 [range 1.6–14.1 vs. 1.1–8.0] × 106 CD34+ cells /kg bw; Pu2009<u20090.05), and also more CD34+ cells per leukapheresis procedure (Pu2009<u20090.001). Consequently, they required a significantly lower number of leukapheresis procedures to achieve the collection goal (median 2.0 vs. 4.0 [range 2–3 vs. 2–9] procedures; Pu2009<u20090.001). The efficiency of the collected stem cells in terms of hematologic engraftment after ABSCT was found to be equal in both groups. These data demonstrate that rescue mobilization with plerixafor triggered by a low stem cell yield in the first leukapheresis session is effective. Although the actual economic benefit may vary depending on the local leukapheresis costs, the median saving of two leukapheresis procedures offsets most of the expenses for the substance in this setting. An exemplary cost calculation is provided to illustrate this effect. J. Clin. Apheresis 29:299–304 2014.

Comparison between intermittent and continuous spectra optia leukapheresis systems for autologous peripheral blood stem cell collection.

Terumo BCT recently introduced a new system for mononuclear cell (MNC) collection that uses a Spectra Optia apheresis machine equipped with a redesigned disposable kit and software program (version 11.2). It allows for the continuous collection of MNCs, unlike the original Spectra Optia system (version 7.2), which included a chamber for two‐step cell separation. The aim of this study was to compare the two apheresis systems in regard to specific performance parameters. A retrospective data analysis of 150 patients who had undergone peripheral blood stem cell collection between March of 2014 and May of 2015 at our institution was performed. For the matched comparison, patients were divided into two groups by diagnosis and by previous forms of therapy received: a homogeneous group of patients with multiple myeloma (MM) that had received first line therapy (“MM” group, nu2009=u200988) and a heterogeneous group that included all of the other patients (“other” group, nu2009=u200962). No significant differences in CD34+ collection yields between both collection regimens were found (pMMu2009=u20090.19, potheru2009=u20090.74) in either group. Moreover, similar performance ratios (collected/predicted CD34+ cell number in %) were observed (pMMu2009=u20090.89, potheru2009=u20090.1). No relevant variations in platelet or hemoglobin loss were found between the two systems. We conclude that the new continuous Spectra Optia MNC system is equally efficient in collecting CD34+ cells and can be used without sacrificing collection efficiency levels when treating a broad variety of autologous patients. J. Clin. Apheresis 32:27–34, 2017.

Performance assessment and benchmarking of autologous peripheral blood stem cell collection with two different apheresis devices

Collection of peripheral blood stem cells (PBSCs) for autologous transplantation is a well‐established process. As a new generation of leukapheresis (LP) machines has been launched, measures of benchmarking and quality control need to be defined in order to ensure consistent collection performance.

Mobilization of autologous and allogeneic peripheral blood stem cells for transplantation in haematological malignancies using biosimilar G-CSF.

Biosimilars of the granulocyte colony stimulating factor (G‐CSF) filgrastim were approved by the European Medicines Agency (EMA) for registered indications of the originator G‐CSF, including prevention and treatment of neutropenia, as well as mobilization of peripheral blood stem cells in 2008. Nevertheless, there is still an ongoing debate regarding the quality, efficacy and safety of biosimilar G‐CSF.

Comparison of biosimilar filgrastim, originator filgrastim, and lenograstim for autologous stem cell mobilization in patients with multiple myeloma

Granulocyte–colony‐stimulating factor (G‐CSF) originators such as filgrastim (Neupogen) and lenograstim (Granocyte) are widely used for peripheral blood stem cell (PBSC) mobilization. In recent years, biosimilar agents have been approved for the same indications. The aim of this retrospective study was to compare the mobilization efficiency of the three G‐CSF variants originator filgrastim, lenograstim, and the biosimilar Filgrastim Hexal in a homogeneous group of multiple myeloma (MM) patients in first‐line therapy.

T cell-based targeted immunotherapies for patients with multiple myeloma

Despite high‐dose chemotherapy followed by autologs stem‐cell transplantation as well as novel therapeutic agents, multiple myeloma (MM) remains incurable. Following the general trend towards personalized therapy, targeted immunotherapy as a new approach in the therapy of MM has emerged. Better progression‐free survival and overall survival after tandem autologs/allogeneic stem cell transplantation suggest a graft versus myeloma effect strongly supporting the usefulness of immunological therapies for MM patients. How to induce a powerful antimyeloma effect is the key issue in this field. Pivotal is the definition of appropriate tumor antigen targets and effective methods for expansion of T cells with clinical activity. Besides a comprehensive list of tumor antigens for T cell‐based approaches, eight promising antigens, CS1, Dickkopf‐1, HM1.24, Human telomerase reverse transcriptase, MAGE‐A3, New York Esophageal‐1, Receptor of hyaluronic acid mediated motility and Wilms tumor gene 1, are described in detail to provide a background for potential clinical use. Results from both closed and on‐going clinical trials are summarized in this review. On the basis of the preclinical and clinical data, we elaborate on three encouraging therapeutic options, vaccine‐enhanced donor lymphocyte infusion, chimeric antigen receptors–transfected T cells as well as vaccines with multiple antigen peptides, to pave the way towards clinically significant immune responses against MM.

Streptamer versus tetramer-based selection of functional cytomegalovirus-specific T cells

BACKGROUND/PURPOSEnCytomegalovirus (CMV) disease constitutes a serious complication after stem cell transplantation and has been treated by adoptive transfer of donor-derived CMV-specific CD8(+) T cells. CMV-specific CD8(+) T cells were selected by multimers, and the technologies may alter the function of these T cells. Therefore, here we evaluated the impact of multimer reagents on the function of CD8(+) T lymphocytes.nnnMETHODSnCMV-specific CD8(+) T cells were purified from the peripheral blood of donors using tetra- and streptamer technologies. The functional status of purified CMV-specific CD8(+) T cells was assessed by multiparametric immunophenotyping and carboxyfluorescein succinimidyl ester proliferation assays as well as by enzyme-linked immunospot assays.nnnRESULTSnA similar percentage of CMV-specific CD8(+) T cells could be purified by both tetra- (90%) and streptamer (92%) technologies. That constitutes a 30- to 50-fold concentration of CMV-specific CD8(+)CD45RA(+)CCR7-effector T cells. Selected cells secreted interferon-gamma and granzyme B upon stimulation with CMVpp65 peptide, thus demonstrating their functionality.nnnCONCLUSIONnOur study demonstrated that both tetra- and streptamer technologies can be used to purify CMV-specific cytotoxic CD8(+) T cells for adoptive T-cell transfer. Both multimer technologies did not have any negative influence on the proliferation of selected T cells. Importantly, streptamer technology is available at good manufacturing practice level.

Standardization of cryopreserved peripheral blood mononuclear cells through a resting process for clinical immunomonitoring—Development of an algorithm

Flow cytometry, as a powerful tool for immunomonitoring and quality control of peripheral blood mononuclear cells (PBMCs), is routinely used in clinical studies. However, flow cytometry based assays for cryopreserved peripheral blood mononuclear cells (cPBMCs) constitute a challenge. Down‐regulation of surface and intracellular markers, as well as impairment of cell function might result from cryopreservation. Furthermore, protocols for resting cPBMCs are available but diverse. Therefore, we performed a standardization of the resting process concerning resting position, cell concentration, resting period and material of cell culture tubes as well as culture media. We further investigated the influence of resting on the phenotype and functionality of T cells comparing fresh PBMCs as gold standard to rested and non‐rested cPBMCs. Polychromatic flow cytometry staining, peptide‐MHC class I restricted tetramer staining and intracellular cytokine staining as major methods were used. Our results revealed that a horizontal position, a cell concentration of 2 to 5 × 106u2009cells/ml and an overnight resting phase is beneficial to eliminate dead or dying cells in cPBMCs with a mean cell loss of 14% overall cell populations. In addition, the quality and quantity of regulatory T cells and antigen specific T cells recovered upon resting. For multifunctional T cells a decrease of activation threshold in the way of a twofold mean fluorescence intensity (MFI) and increase of degranulation marker CD107a, co‐stimulatory marker CD28, adhesion molecule CD62L as well as the ability to secrete antiviral cytokines like interferon gamma (IFN‐γ), tumor necrosis factor (TNF), and interleukin 2 (IL‐2) comparable to fresh PBMCs were observed. However, based upon our data resting is not helpful for the flow cytometric analyses of myeloid‐derived suppressor cells (MDSCs) and large/intermediate size lymphocytes which rather decreased/vanished ex vivo. Therefore, we developed an algorithm to indicate for which cell population and for which type of analyses the resting process is useful or not.