Poster B1 - Investigating the Critical Process Parameters of Therapeutic T Cell Manufacturing Using Degradable Microscaffold Activation

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Viviana Velez

Chemical Engineering Undergraduate
University of Puerto Rico, Mayaguez
Isabela, Puerto Rico, United States

Co-Author(s)

MA

Miguel Armenta, MS

PhD Candidate
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
Atlanta, Georgia, United States

Primary Investigator(s)

KR

Krishnendu Roy

Dean of Engineering
Vanderbilt University, United States

Introduction: T cell activation is a complex process requiring extracellular signals mediated by T cell receptor (TCR) complexes. This complexity translates to in-vitro expansion, where specific monoclonal antibodies provide the necessary signals for activation, which is crucial in developing effective immunotherapies. While T-cell therapies show great promise in treating various diseases, there are significant challenges in their manufacturing, including limited cell expansion platforms and the need for standardized methods capable of producing high-quality therapeutic cells. This project aims to develop a scalable T-cell manufacturing platform using novel antibody-functionalized degradable microscaffolds (DMSs). These DMSs can mimic the high-density cellular microenvironment experienced by T cells in-vivo and have been shown to improve T cell memory profiles across multiple donors. Overall, this work seeks to optimize T cell expansion and therapeutic quality by investigating the critical process parameters in their manufacturing.

Materials and

Methods: The approach begins with the DMS fabrication where collagen-derived gelatine microcarriers (Cultispher-S, GE Healthcare) are prepared by hydrating and autoclaving them to ensure sterility. Then, they are coated in sulfo-NHS-biotin (ThermoFisher) followed by Streptavidin (Jackson ImmunoResearch). Finally, they are functionalized by coating them in biotinylated monoclonal antibodies against CD3 and CD28 (Biolegend) or any other combinations to be tested. All the coating steps were quantified to ensure quality control between lots. The finished DMSs are then used to culture either Peripheral Mononuclear Cells (PBMCs) or isolated T cells from matched donors (AllCells, Isolation kit Miltenyi Biotec) with recombinant IL-2 (Peprotech) in serum-free OpTmizer medium (ThermoFisher) for six to eight days. After 48 hours of culture, the cells are transduced to express an anti-BCMA CAR (Plasmid from NIH). Glucose and lactate levels are monitored daily (ChemGlass, Nova Biomedical) after transduction to ensure process control leading to a more stable and predictable culture environment. Media addition is based on daily glucose readings adding media for readings below 250 mg/dL threshold. Finally, cells are harvested after DMSs degradation by adding 0.5mg/mL Collagenase D (Sigma Aldrich) and incubating for about fifteen minutes. Cell counts and viability are assessed during the harvest. Spectral flow cytometry is performed after harvesting using a ~20-color panel to quantify the phenotypic distribution of the culture regarding memory and exhaustion. These data are then compared to day zero to determine changes driven by the tested culture conditions.

Results, Conclusions, and Discussions: In testing isolated vs. un-isolated cells in culture with DMSs or TransAct (Miltenyi Biotec), glucose was monitored daily as presented in Figure A to track any differences in consumption rates. TransAct was the first activator to reach the media addition threshold regardless of isolation. DMS E did not reach the threshold until harvest, but again both isolation conditions behaved similarly. Conversely, DMSs J and K required media additions at the same time as TransACT, but only in the un-isolated cultures. Figure B validates that glucose consumption is due to T cell proliferation and not metabolic reprogramming, as fold expansion was significantly higher for DMSs J and K in unisolated cultures.

Figure C looks at the phenotypic distribution of the manufactured cells. For isolated cultures, CD4+ cell counts were significantly higher; while for un-isolated cultures, CD8+ counts were greater. We hypothesize that the early presence of other cell types in the PBMCs results in paracrine signaling that promotes CD8+ expansion. However, when comparing memory subtype distributions with and without isolation, no significant differences were observed. As expected, central memory is shown to be the dominant subtype; however, there is a sizable naïve and stem cell memory population. These subtypes have been key manufacturing targets as their high proliferative capacity and long-term persistence make them particularly valuable for therapeutic settings. Interestingly, Figure D shows significant differences in Naïve subtype counts based on the activator used. In general, all DMSs tested did a better job at expanding Naïve T cells than the commercial activator TransAct. Furthermore, we can observe an inverse relation between antibody concentration and the Naïve population reaching a greater cell count with DMS K independent of isolation.

In conclusion, DMSs can improve T cell memory profiles which have been associated with better patient outcomes. Furthermore, their easy degradation streamlines the cell harvesting process, reducing time and labor costs, thereby increasing efficiency. Additionally, improvements in DMS manufacturing quality control have resulted in less variability allowing for better experimental control during the T cell expansions. These innovations collectively represent a transformative step towards achieving more consistent, effective and scalable cell therapy manufacturing.

Acknowledgements (Optional): This material is based upon work supported by the National Science Foundation under Grant No. EEC-1648035.