#28-Cell and Gene Therapy Today
Stay up to date with the latest developments in your field with our weekly digest of industry news and research articles.
I’m Pedro Silva Couto, and this is Cell and Gene Therapy Today. Here I will be sharing of the most recent news in CGT field as well as summarizing research articles focused on translational research, manufacturing and clinical studies featuring cell and gene therapy product candidates.
CGT News this week:
🧱 Lyell Immunopharma to partner with Cellares to assess its automated manufacturing platform, the Cell Shuttle™, for producing Lyell’s LYL797 CAR T-cell therapy (news here). This product targets ROR1 protein and is aimed at solid tumors. Lyell’s pipeline features two CAR-T products and two TIL product candidates at the moment.
📈 MHRA approves Rentschler Biopharma's Stevenage plant for clinical production of gene therapy vectors (news here). This manufacturing site has received cGMP manufacturing compliance certification and should be primarily used to produce adeno-associated virus (AAV) vectors.
📰 US FDA approves plan to resolve clinical hold on HEMO-CAR-T IND from Hemogenyx Pharma started in June 2023 (news here). The regulator’s concerns, related to a splicing issue during lentivirus manufacturing, have been addressed, paving the way for the resumption of clinical trials. The company has now identified the source of the splicing issue and created a way to eliminate it.
🧱 The CDMO Fujifilm Diosynth is close to opening a cutting-edge facility in the UK (Darlington) to cope with the demand from the advanced therapy manufacturing space (news here). This facility specializes in guiding clients through the transition from pre-clinical stages to Phase I programs, with a special emphasis on advancing therapies such as gene therapies, oncolytic viruses, and viral vaccines.
📈 Abu Dhabi Stem Cells Centre makes history with UAE's first CAR-T cell treatment for leukaemia in 11-year-old patients (news here). The company’s pipeline include other product candidates such as hematopoietic stem cells to target multiple sclerosis or mesenchymal stromal cells delivered with an extra-corporeal device.
CGT Research this week:
CAR-T manufacturing and clinical outcome
Study focused on the expansion part of the CAR-T manufacturing process has demonstrated that prolonged in vitro culture periods were associated to increase DNA methylation levels and subsequently reduced long-term survival post transfusion (study here).
After a recent publication from this group suggesting that methylation events induced during transduction were associated with lower efficacy of the CAR-T treatment, this study focuses on the expansion part of the process and its impact on the overall product quality. This study expanded transduced and non-transduced T-cells during 22 days using different mechanisms and evaluated their growth kinetics, immunophenotype performing DNA methylation and gene expression studies as well. Similarly to several other studies, the authors reported that IL-2 promoted faster growth kinetics compared to IL-7/IL-15 combination and that multiple activation cycles further enhanced the expansion. However, this happened at the expense of continuous and pronounced DNA methylation changes in particular to genes related to T-cell function. The authors conducted a gene expression analysis between seeding and harvesting (D0 versus D22) and concluded that the expansion per se led to the downregulation of genes involved in T-cell activation, differentiation and alpha-beta T-cell activation. Given its role in promoting T-cell proliferation, IL-2 was associated with extended proliferation and therefore DNA methylation levels. The authors then analyzed patient material from the clinical trials NCT03144583, NCT02772198 and NCT03373071 and concluded that DNA methylation levels were associated with lower overall survival. At the end of the study the authors remained however cautious and afirmed that although expansion time was associated with higher DNA methylation levels, it remains unclear whether very short culture times lead to improved clinical outcomes. Regarding the manufacturing processes used, CD3 selection was performed from PBMCs using MicroBead kits ) followed by the activation step performed using CD2/CD3/CD28-loaded beads or CD3/CD28 TransAct (Miltenyi). Cytokine supplementation was ensured via IL-2 (20 ng/mL) or IL-7/IL-15 addition (12.5 ng/mL) depending on the activation chosen. The expansion cycle was performed using TexMACS (Miltenyi) supplemented with 3% AB serum and the previously described cytokine supplementation.
Cord blood as a source for allogeneic CAR-T
Manufacturing study demonstrated feasibility of using cord blood as starting material for a CAR-T product exhibiting in vitro and in vivo killing capabilities (study here).
This study puts forward cord blood to serve the allogeneic CAR-T manufacturing space given its usage in the hematological malignancies field as an alternative to bone marrow. Prior studies have hypothesized that this perinatal cell source may lead to improved anti-cancer efficacy resulting in unique biological properties not present in PBMCs. In this work, the authors started by demonstrating that CD3+ cells were successfully isolated from cord blood and transduced using a lentiviral vector. This process did not result in immunophenotypic changes in the CD4:CD8 and the cytotoxic T cell phenotype fractions. An RNA-seq analysis demonstrated that the transduction step did change the expression of genes associated with the cytokine-cytokine receptor interactions, chemokine signaling pathways and antigen presentation. The authors went on to demonstrate the cytotoxicity capabilities of the CB-derived CAR-T product on the BV173 cell line (CD19 target) keeping K562 as a negative control. The in vivo studies conducted demonstrated product efficacy using a mouse model and further increased the evidence on the specificity of the killing effect upon activation from the target CD19 populations. This was confirmed by the significantly higher secreted levels of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ in the CAR product administration but not from the non-transduced control. The manufacturing process used in this work featured CD3 positive isolation using microbead technology (Miltenyi). While activation was delivered using TransAct (Miltenyi) the medium formulation used throughout the process was XVIVO-15 (Lonza) supplemented with 5% AB serum (with 200 IU/mL of IL-2 (PrepoTech). Transduction was conducted using 24 well plates pre-coated with retronectin at the concentration of 6 µg/cm2 and a spinnocoulation cycle.
iPSC-derived CAR-NK Production
Manufacturing study puts forward a workflow featuring both electroporation and viral transduction to generate iPSCs and CAR-iPSCs respectively, using differentiation as last step before demonstrating functionality of the iCAR-NK product (study here).
In this study, the authors put forward CAR-NK products as an alternative to CAR-T, given their lower risk associated with treatment-induced GvHD, acute cytokine release syndrome and neurotoxicity. In this study, the authors used iPSCs to avoid some of the bottlenecks associated with CAR-NK: (1) low frequency when isolated from PBMCs and (2) low transduction efficiencies that NK typically report when infected with viral vectors.
The authors started by describing that the isolation of CD56+ cells was performed using FACS, whereas the reprogramming step was delivered using three episomal plasmids coding for SOX2, KLF4, L-MYC, LIN28, OCT3/4 and shRNA against p53, and transient EBNA-1 was delivered via electroporation (4D nucleofector, Lonza). Transduction to introduce the anti-CD19 CAR transgene was performed in the presence of Vectofusin-1 (Miltenyie) at the concentration of 10 μg/mL using a spinnoculation cycle performed at 400g for 2h. The authors then proceeded with single-clone isolation to select an iPSC-CAR candidate. The differentiation into CAR-NK cells was achieved using a two-step protocol: (1) hematopoietic progenitor formation (CD34+) enabled via embryoid body formation and (2) selection of the resulting suspension fraction and subculture with a cocktail of defined cytokines (SCF, IL-7, IL-15, IL-3 and Flt ligand). The authors then proceeded to demonstrate superior cytotoxicity of the i-CAR-NK product compared to CAR-iPSCs when co-cultured with Raji, REH target cells and K562 (CD19 negative). Given that the authors observed killing of the K562 cell line, the hypothesis was that the iCAR-NK product may have CAR-independent cytotoxicity. Summarizing the Pros and Cons of this manufacturing approach:
Pros
Generated a CAR-iPSC bank that can be used for multiple applications (NK, T).
Started with a widely available and healthy cell source.
Put forward electroporation as a low-cost and scalable approach to generate iPSCs.
Cons
Some of the steps required are hardly scalable (EB formation, NK differentiation and FACS).
The differentiation step requires a very defined cytokine cocktail composition and can take up to 35 days.
Due to the cytokine-rich process, process costs based on this strategy may have reagent-associated high costs.
This is it for this week’s Cell and Gene Therapy Today, which I aim to send you every Wednesday. If you found it valuable, please feel free to sign up or consider sending it to someone who finds this content useful!
I am currently a post-doctoral researcher at the University College London, and my project is focused on scalable CAR-T cell manufacturing using non-viral methods. You can find more about my research on my Google Scholar or my LinkedIn page.
Last but not least, this content was only possible to produce with the sponsorship of celltrials.org, the leading online portal tracking the clinical trial landscape of cell and gene therapy products. They have data packages on CAR-T, hMSCs, Extracellular Vesicles, and Cell-Free products, as well as in vivo gene therapy products (visit celltrials.org).