Scott R. Burger

HPC viability measurement : trypan blue versus acridine orange and propidium iodide

BACKGROUND: A reliable, validated method for rapidly determining HPC viability is essential for clinical cell engineering.

Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells.

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interactionin vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70–90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15–25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.

Potency assay development for cellular therapy products: an ISCT∗ review of the requirements and experiences in the industry

The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development.

Developing assays to address identity, potency, purity and safety: cell characterization in cell therapy process development

A major challenge to commercializing cell-based therapies is developing scalable manufacturing processes while maintaining the critical quality parameters (identity, potency, purity, safety) of the final live cell product. Process development activities such as extended passaging and serum reduction/elimination can facilitate the streamlining of cell manufacturing process as long as the biological functions of the product remain intact. Best practices in process development will be dependent on cell characterization; a thorough understanding of the cell-based product. Unique biological properties associated with different types of cell-based products are discussed. Cell characterization may be used as a tool for successful process development activities, which can promote a candidate cell therapy product through clinical development and ultimately to a commercialized product.

Clinical-scale selection of anti-CD3/CD28-activated T cells after transduction with a retroviral vector expressing herpes simplex virus thymidine kinase and truncated nerve growth factor receptor

Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.

Concise Review: Guidance in Developing Commercializable Autologous/Patient-Specific Cell Therapy Manufacturing

Cell therapy is poised to play an enormous role in regenerative medicine. However, little guidance is being made available to academic and industrial entities in the start‐up phase. In this technical review, members of the International Society for Cell Therapy provide guidance in developing commercializable autologous and patient‐specific manufacturing strategies from the perspective of process development. Special emphasis is placed on providing guidance to small academic or biotech researchers as to what simple questions can be addressed or answered at the bench in order to make their cell therapy products more feasible for commercial‐scale production. We discuss the processes that are required for scale‐out at the manufacturing level, and how many questions can be addressed at the bench level. The goal of this review is to provide guidance in the form of topics that can be addressed early in the process of development to better the chances of the product being successful for future commercialization.

Design and operation of a current good manufacturing practices cell-engineering laboratory

Medical centers and biotechnology companies active in cellular and gene therapy increasingly are working to design and build clinical laboratories capable of performing cellular engineering and vector production using current good manufacturing practices (cGMPs). Because cell engineering is a rapidly changing field, and definitions for cell engineering cGMPs are still being established, a cGMP cell-engineering laboratory most often should be designed with a broad range of potential applications in mind. While the laboratory facility is the most tangible aspect of cGMP, it represents only part of a larger process, which it must be designed and built to support.

Validation of short-term handling and storage conditions for marrow and peripheral blood stem cell products

BACKGROUND: Allogeneic hematopoietic stem cell transplants from unrelated donors are routinely used in the treatment of patients with hematologic malignancies. These cellular products are often collected off‐site and require transport from the collection site to transplantation centers. However, the effects of transport conditions and media on stem cell graft composition during short‐term storage have not been well described.

Retroviral transduction and expansion of peripheral blood lymphocytes for the treatment of mucopolysaccharidosis type II, Hunter's syndrome.

BACKGROUND: Gene therapy using autologous peripheral blood lymphocytes (PBLs) has been used to produce adenosine deaminase with which to treat patients with severe combined immunodeficiency. Patients with mucopolysaccharidosis type II (MPS II) lack iduronate‐2‐sulfatase (IDS), and serial PBL gene therapy may benefit these patients.

Improved progenitor assay standardization using peripheral blood progenitor cells from a donor treated with granulocyte–colony‐stimulating factor

BACKGROUND: Progenitor assays are the principal method for evaluating hematopoietic cell function. The magnitude of assay variability and the assay steps contributing to variability were determined, and modifications intended to increase assay consistency were evaluated.