Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells. METHODS: We used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency. RESULTS: Results indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells. CONCLUSIONS: These studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain. METHODS: Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model. RESULTS: In comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model. CONCLUSIONS: This study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.

Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

A multiscale simulation framework for the manufacturing facility and supply chain of autologous cell therapies.

BACKGROUND AIMS: Autologous cell therapy (AuCT) is an emerging therapeutic treatment that is undergoing transformation from laboratory- to industry-scale manufacturing with recent regulatory approvals. Various challenges facing the complex AuCT manufacturing and supply chain process hinder the scale out and broader application of this highly potent treatment. METHODS: We present a multiscale logistics simulation framework, AuCT-Sim, that integrates novel supply chain system modeling algorithms, methods, and tools. AuCT-Sim includes a single facility model and a system-wide network model. Unique challenges of the AuCT industry are analyzed and addressed in AuCT-Sim. Decision-supporting tools can be developed based on this framework to explore "what-if" manufacturing and supply chain scenarios of importance to various cell therapy stakeholder groups. RESULTS: Two case studies demonstrate the decision-supporting capability of AuCT-Sim where one investigates the optimal reagent base stocking level, and the other one simulates a reagent supply disruption event. These case studies serve as guidelines for designing computational experiments with AuCT-Sim to solve specific problems in AuCT manufacturing and supply chain. DISCUSSION: This simulation framework will be useful in understanding the impact of possible manufacturing and supply chain strategies, policies, regulations, and standards informing strategies to increase patient access to AuCT.

The potential of CAR T therapy for relapsed or refractory pediatric and young adult B-cell ALL.

Recent advancements in immunooncology have resulted in the generation of novel therapies such as chimeric antigen receptor (CAR) T cells, which have revolutionized the treatment of pediatric patients with relapsed or refractory B-cell acute lymphoblastic leukemia. The journey of tisagenlecleucel (formerly CTL019) from early preclinical success to the US Food and Drug Administration approval is summarized in this review. Strategies that are currently being investigated to improve the efficacy and safety profile of CAR T-cells are also explored, as well as the factors contributing to the present state of patient access to CAR T therapy.

Bioengineering Solutions for Manufacturing Challenges in CAR T Cells.

The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed "living drugs" for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly developed into powerful tools to harness the power of immune system manipulation against cancer. Regulatory agencies are beginning to approve CAR T cell therapies due to their striking efficacy in treating some hematological malignancies. However, the engineering and manufacturing of such cells remains a challenge for widespread adoption of this technology. Bioengineering approaches including biomaterials, synthetic biology, metabolic engineering, process control and automation, and in vitro disease modeling could offer promising methods to overcome some of these challenges. Here, we describe the manufacturing process of CAR T cells, highlighting potential roles for bioengineers to partner with biologists and clinicians to advance the manufacture of these complex cellular products under rigorous regulatory and quality control.

Epidermal growth factor mediated healing in stem cell-derived vocal fold mucosa.

BACKGROUND: The goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. MATERIALS AND METHODS: A scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation after injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, gefitinib. RESULTS: Wound repair after injury was significantly hastened by application of exogenous EGF (13.3 µm/h, ± 2.63) compared with absence of exogenous EGF (7.1 µm/h ± 2.84), but inhibited with concurrent addition of Gefitinib (5.2 µm/h, ± 2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. Although not statistically significant, increased density of Ki67 staining in the epithelium adjacent to the scratch wound was observed after treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. CONCLUSIONS: Exogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair after injury.

Temporal impact of substrate mechanics on differentiation of human embryonic stem cells to cardiomyocytes.

A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces, which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes.

Improving efficiency of human pluripotent stem cell differentiation platforms using an integrated experimental and computational approach.

Human pluripotent stem cells (hPSCs) have an unparalleled potential for tissue engineering applications including regenerative therapies and in vitro cell-based models for studying normal and diseased tissue morphogenesis, or drug and toxicological screens. While numerous hPSC differentiation methods have been developed to generate various somatic cell types, the potential of hPSC-based technologies is hinged on the ability to translate these established lab-scale differentiation systems to large-scale processes to meet the industrial and clinical demands for these somatic cell types. Here, we demonstrate a strategy for investigating the efficiency and scalability of hPSC differentiation platforms. Using two previously reported epithelial differentiation systems as models, we fit an ODE-based kinetic model to data representing dynamics of various cell subpopulations present in our culture. This fit was performed by estimating rate constants of each cell subpopulation's cell fate decisions (self-renewal, differentiation, death). Sensitivity analyses on predicted rate constants indicated which cell fate decisions had the greatest impact on overall epithelial cell yield in each differentiation process. In addition, we found that the final cell yield was limited by the self-renewal rate of either the progenitor state or the final differentiated state, depending on the differentiation protocol. Also, the relative impact of these cell fate decision rates was highly dependent on the maximum capacity of the cell culture system. Overall, we outline a novel approach for quantitative analysis of established laboratory-scale hPSC differentiation systems and this approach may ease development to produce large quantities of cells for tissue engineering applications.