Mechanism of action, potency and efficacy: considerations for cell therapies.

One of the most challenging aspects of developing advanced cell therapy products (CTPs) is defining the mechanism of action (MOA), potency and efficacy of the product. This perspective examines these concepts and presents helpful ways to think about them through the lens of metrology. A logical framework for thinking about MOA, potency and efficacy is presented that is consistent with the existing regulatory guidelines, but also accommodates what has been learned from the 27 US FDA-approved CTPs. Available information regarding MOA, potency and efficacy for the 27 FDA-approved CTPs is reviewed to provide background and perspective. Potency process and efficacy process charts are introduced to clarify and illustrate the relationships between six key concepts: MOA, potency, potency test, efficacy, efficacy endpoint and efficacy endpoint test. Careful consideration of the meaning of these terms makes it easier to discuss the challenges of correlating potency test results with clinical outcomes and to understand how the relationships between the concepts can be misunderstood during development and clinical trials. Examples of how a product can be "potent but not efficacious" or "not potent but efficacious" are presented. Two example applications of the framework compare how MOA is assessed in cell cultures, animal models and human clinical trials and reveals the challenge of establishing MOA in humans. Lastly, important considerations for the development of potency tests for a CTP are discussed. These perspectives can help product developers set appropriate expectations for understanding a product's MOA and potency, avoid unrealistic assumptions and improve communication among team members during the development of CTPs.

Sepax-2 cell processing device: a study assessing reproducibility of concentrating thawed hematopoietic progenitor cells.

BACKGROUND: Autologous hematopoietic progenitor cell (HPC) transplantation is currently the standard of care for a fraction of patients with newly diagnosed myelomas and relapsed or refractory lymphomas. After high-dose chemotherapy, cryopreserved HPC are either infused directly after bedside thawing or washed and concentrated before infusion. We previously reported on the comparability of washing/concentrating HPC post-thaw vs. infusion without manipulation in terms of hematopoietic engraftment, yet settled for the prior favoring cell debris and DMSO removal. For almost two decades, automation of this critical step of washing/concentrating cells has been feasible. As part of continuous process verification, we aim to evaluate reproducibility of this procedure by assessing intra-batch and inter-batch variability upon concentration of thawed HPC products using the Sepax 2 S-100 cell separation system. METHODS: Autologous HPC collected from the same patient were thawed and washed either in two batches processed within a 3-4 h interval and immediately infused on the same day (intra-batch, n = 45), or in two batches on different days (inter-batch, n = 49) for those patients requiring 2 or more high-dose chemotherapy cycles. Quality attributes assessed were CD34+ cell recovery, viability and CD45+ viability; CFU assay was only performed for allogeneic grafts. RESULTS: Intra-batch and inter-batch median CD34+ cell recovery was comparable (75% vs. 73% and 77% vs. 77%, respectively). Similarly, intra-batch and inter-batch median CD45+ cell viability was comparable (79% vs. 80% and 79% vs. 78%, respectively). Bland-Altman analysis describing agreement between batches per patient revealed a bias close to 0%. Additionally, lower HPC recoveries noted in batch 1 were noted as well in batch 2, regardless of the CD34+ cell dose before cryopreservation, both intra- and inter-batch, suggesting that the quality of the collected product plays an important role in downstream recovery. Intrinsic (high mature and immature granulocyte content) and extrinsic (delay between apheresis and cryopreservation) variables of the collected product resulted in a significantly lower CD45+ viability and CD34+ cell recovery upon thawing/washing. CONCLUSIONS: Automated post-thaw HPC concentration provides reproducible cell recoveries and viabilities between different batches. Implications of this work go beyond HPC to concentrate cell suspension/products during manufacturing of cell and gene therapy products.

Nonclinical and quality assessment of cell therapy products: Report on the 4th Asia Partnership Conference of Regenerative Medicine, April 15, 2021.

The 4th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 15, 2021, to promote regulatory harmonization of regenerative medicine products throughout Asia. Recognizing domestic regulatory guidelines within each country and region, and their underpinning rationales, is an important initial step toward a convergence of regulations. The 4th APACRM consisted of an open dialog with regulatory agencies regarding nonclinical and quality settings for cell therapy products (CTPs) through industry presentations and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region, and specific regulatory schematics in Japan, were also introduced. The objective of this paper is to summarize the proceedings of the 4th APACRM for public dissemination and to foster further discussion in the future.

Non-clinical assessment of cell therapy products: the perspective from five Asian countries/regions based on regulatory guidelines and the underpinning rationales.

BACKGROUND AIMS: Cell-based regenerative medicine is an innovative field that can potentially alter the overall survival and quality of life of patients with devastating diseases. Several cell therapy products (CTPs) have been approved within the last two decades, and more are under development. The establishment of an effective developmental strategy in accordance with the regulatory bodies of each country/region is crucial for fast delivery of each respective CTP. In particular, facilitating investigational new drug (IND) approval is important for accelerating the transition from non-clinical to clinical research/trial phases. METHODS: Here the authors compared the non-clinical prerequisites for initiating clinical studies in five Asian countries/regions (India, China, Korea, Taiwan and Japan) from an industry viewpoint. The authors first identified the differences and tried to clarify the perspectives/considerations underpinning the different requirements. RESULTS: The authors' findings revealed that differences in regulations and development experiences, especially with CTPs, have led to clear differences in the non-clinical study package and its corresponding study design. CONCLUSIONS: By sharing experiences of the research and development of CTPs among Asian countries/regions and including not only industry but also regulatory authorities, we will be able to expedite cross-border IND approval and eventually contribute to the early delivery of innovative CTPs to many Asian patients.

Validation of an automated blood culture system for sterility testing of cell therapy products.

BACKGROUND AIMS: Automated blood culture systems are widely used for the detection of microorganisms in cell therapy products. However, they are not validated by the manufacturers for this purpose. The aim of this study was to assess the ability of the Bactec system (Becton-Dickinson, Le Pont-De-Claix, France) to detect the microorganisms that could contaminate cell therapy products. METHODS: Three types of vials and conditions were tested: Plus Aerobic/F and Anaerobic/F media incubated at 35°C and Mycosis IC/F medium incubated at 30°C. All vials were incubated 10 days. We tested 18 microorganisms, including slow growers and some with fastidious nutritional requirements (10 bacteria, four yeasts, four filamentous fungi), each with four inocula (10-10(4) colony-forming units) performed in quintuplicate. RESULTS: The combination of Plus Aerobic/F and Plus Anaerobic/F vials detected all the tested pathogenic bacteria, all the tested Gram-positive skin commensal or environmental bacteria, all the tested yeasts, and three of four tested filamentous fungi. The addition of the Mycosis IC/F vial extended the range of detected microorganisms to one fungal environmental contaminant. Two bacterial environmental contaminants were not detected by our method. Low inocula of the skin contaminant Propionibacterium acnes were detected only after 7 days of incubation. CONCLUSIONS: These data suggest that (i) the prolongation of the incubation time of Plus Aerobic/F and Plus Anaerobic/F vials from 7 to 10 days and (ii) the use of Mycosis IC/F medium make minor contributions in the sterility testing of cell therapy products. We have validated the Bactec method using aerobic and anaerobic vials incubated 7 days at 35°C.

Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/µg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

Cryoprotection in Human Mesenchymal Stromal/Stem Cells: Synergistic Impact of Urea and Glucose.

Standard freezing protocols of clinically relevant cell lines commonly employ agents such as fetal bovine serum and dimethyl sulfoxide, which are a potential concern from both a regulatory and a patient safety perspective. The aim of this work was to develop formulations with safe and well tolerated excipients for the (cryo-) preservation of cell therapy products. We evaluated the cryoprotective capabilities of urea and glucose through measurements of cell metabolic activity. Freezing of clinically relevant human mesenchymal stromal/stem cells and human dermal fibroblasts at ≤ - 65°C at equimolar ratios of urea and glucose resulted in comparable viabilities to established dimethyl sulfoxide. Pre-incubation of human mesenchymal stromal/stem cells in trehalose and addition of mannitol and sucrose to the formulation further enhanced cell viability after freeze-thaw stress. Other cell types assessed (A549 and SK-N-AS) could not satisfactorily be preserved with urea and glucose, highlighting the need for tailored formulations to sustain acceptable cryopreservation.

Comparison of cell viability methods for human mesenchymal/stromal stem cells and human A549 lung carcinoma cells after freeze-thaw stress.

For the safety and efficacy of frozen cell therapy products, determination of cellular viability is key. However, results of cell viability measurements do not only depend on the cell line or on the inflicted stress, but also on the assay used, making inter-experimental comparisons difficult. The aim of this study was thus to assess commonly used viability assays in clinically relevant human mesenchymal/stromal stem cells and human A549 lung carcinoma cells. Post freeze-thaw stress viability and proliferation were evaluated under different conditions using trypan blue, acridine orange/DAPI stain, alamarBlue, ATP, and neutral red assays. Significant differences in cell viability between metabolic assays were observed, likely due to their distinct intrinsic detection mechanisms. Membrane-integrity based assays generally overestimated cell viabilities in this study. Furthermore, noticeable differences in inter-assay sensitivities were observed. These differences highlight that cell viability methods should be meticulously selected and their associated results carefully interpreted in a relevant context to ensure reliable conclusions. Indeed, although cell membrane integrity based assays are a popular choice to determine cellular quality attributes after freezing and thawing, we demonstrate that metabolic assays may be more suitable in this context.

Comparison of Three Methods for Preparation of Autologous Cells for Use in Cell Therapy of Chronic Limb-Threatening Ischemia in People with Diabetes.

Autologous cell therapy (ACT) is a new therapeutic approach for diabetic patients with no-option chronic limb-threatening ischemia (NO-CLTI). The aim of our study was to quantify cell populations of cell therapy products (CTPs) obtained by three different isolation methods and to correlate their numbers with changes in transcutaneous oxygen pressure (TcPO2). CTPs were separated either from stimulated peripheral blood (PB) (n = 11) or harvested from bone marrow (BM) processed either by Harvest SmartPReP2 (n = 50) or sedimented with succinate gelatin (n = 29). The clinical effect was evaluated by the change in TcPO2 after 1, 3 and 6 months. TcPO2 increased significantly in all three methods at each time point in comparison with baseline values (p < .01) with no significant difference among them. There was no correlation between the change in TcPO2 and the size of injected cell populations. We only observed a weak correlation between the number of injected white blood cells (WBC) and an increase in TcPO2 at 1 and 3 months. Our study showed that all three isolation methods of ACT were similarly relatively efficient in the treatment of NO-CLTI. We observed no correlation of TcPO2 increase with the number of injected monocytes, lymphocytes or CD34+. We observed a weak correlation between TcPO2 increase and the number of injected WBCs.

Biodistribution studies for cell therapy products: Current status and issues.

Information on the biodistribution (BD) of cell therapy products (CTPs) is essential for prediction and assessment of their efficacy and toxicity profiles in non-clinical and clinical studies. To conduct BD studies, it is necessary to understand regulatory requirements, implementation status, and analytical methods. This review aimed at surveying international and Japanese trends concerning the BD study for CTPs and the following subjects were investigated, which were considered particularly important: 1) comparison of guidelines to understand the regulatory status of BD studies in a global setting; 2) case studies of the BD study using databases to understand its current status in cell therapy; 3) case studies on quantitative polymerase chain reaction (qPCR) used primarily in non-clinical BD studies for CTPs; and 4) survey of imaging methods used for non-clinical and clinical BD studies. The results in this review will be a useful resource for implementing BD studies.

Single-Cell RNA Sequencing of Human Pluripotent Stem Cell-Derived Macrophages for Quality Control of The Cell Therapy Product.

Macrophages exhibit high plasticity to achieve their roles in maintaining tissue homeostasis, innate immunity, tissue repair and regeneration. Therefore, macrophages are being evaluated for cell-based therapeutics against inflammatory disorders and cancer. To overcome the limitation related to expansion of primary macrophages and cell numbers, human pluripotent stem cell (hPSC)-derived macrophages are considered as an alternative source of primary macrophages for clinical application. However, the quality of hPSC-derived macrophages with respect to the biological homogeneity remains still unclear. We previously reported a technique to produce hPSC-derived macrophages referred to as iMACs, which is amenable for scale-up. In this study, we have evaluated the biological homogeneity of the iMACs using a transcriptome dataset of 6,230 iMACs obtained by single-cell RNA sequencing. The dataset provides a valuable genomic profile for understanding the molecular characteristics of hPSC-derived macrophage cells and provide a measurement of transcriptomic homogeneity. Our study highlights the usefulness of single cell RNA-seq data in quality control of the cell-based therapy products.