Chronic hepatitis B virus (HBV) infection can result in hepatocellular carcinoma (HCC), which is the third leading cause of cancer-related deaths worldwide. For autologous immunotherapy of HCC, patient-derived T cells can be engineered to target HBV-specific antigens by exogenous expression of HBV-specific T cell receptors (TCRs). In contrast to viral delivery methods that can be expensive, time-consuming, inefficient, and potentially toxic during treatment due to the long-lived survival of transduced cells, non-viral mRNA electroporation is a transient, cheaper, and potentially safer alternative. However, high efficiency of transfection, high cell viability, and scalability are important parameters for commercial production of quality, clinically-functional engineered cells. Here, we demonstrate seamless optimization of electroporation conditions for efficient and scalable electroporation of activated primary T cells with HBV-specific TCR mRNA at a scale up to 3.9 billion cells. Moreover, T cells could be cryopreserved soon after electroporation and yielded TCR expression and functional cytokine expression comparable to freshly- electroporated T cells upon thawing. This study demonstrates a robust and efficient manufacturing process for large-scale, transient TCR-expressing T cells with a cGMP-compliant non-viral cell engineering platform.

Workflow Schematic

Electroporation Protocol and Cell Density Optimization

Figure 2: MaxCyte electroporation enables rapid optimization for mRNA transfection of high-cell-density, primary, activated T cells.
5 x 10⁶ primary activated T cells were electroporated with TCR mRNA in an OC-100 processing assembly following 3 T cell-specific protocols: Low, medium or high energy. A) FACS analysis revealed high TCR expression in all 3 transfections. B) 1 x 10⁸ , 2 x 10⁸ and 3 x 10⁸ cells/mL were electroporated with 100 or 200 ug/mL TCR mRNA. FACS analysis demonstrated efficient TCR expression at all 3 cell densities; higher mRNA concentration and cell density led to higher TCR expression.

Scalable Electroporation in Static Processing Assemblies

Figure 3: Primary human activated T cells were electroporated in a scalable manner (15 million to 570 million cells) using MaxCyte static processing assemblies, resulting in comparable and high transfection efficiency, cell viability and cytokine function.
Primary activated T cells were electroporated with TCR mRNA in MaxCyte static processing assemblies (OC100x2, OC400, and CL1.1 corresponding to a total of 1.5 x 10⁷, 6 x 10⁷, and 5.7 x 10⁸ cells, respectively). A) TCR expression (89-91%) in CD8+ and CD4+ T cell populations, and B) cell viability (81-85%) were similar for cells electroporated in all three static PAs. C-D) The day after electroporation, TCR expressing lymphocytes were co-cultured with antigen peptide-pulsed T2 cells and IFNγ-expressing cells were analyzed by flow cytometry. The percentage of IFNγ-expressing cells and Mean Flourescence Intensity (MFI) were similar in T cells electroporated with all three static PAs.

Scalable Efficiency in Static and Flow Electroporation Processing Assemblies

Figure 4: MaxCyte Static and Flow Electroporation results in high electroporation efficiency, viability, and functional cytokine release.
Primary activated T cells were electroporated with TCR mRNA in MaxCyte static (OC-400) or flow (CL-2) processing assemblies (PAs). The total number of T cells electroporated in the OC-400 and CL-2 was 1.2E8 and 3.9E9, respectively. T cells were analyzed 24, 48, and 72 hours after transfection by flow cytometry. A) TCR expression was >90% in both PAs for the first 48 hours and dropped to approximately 50% 72 hours after transfection. B) Cell viability was comparable (82-86%) in both PAs over 72 hours. Furthermore, viability did not change in the absence or presence of mRNA. C-D) TCR expressing lymphocytes were co-cultured with antigen pulsed T2 cells and IFNγ- expressing cells were analyzed by flow cytometry. C) TCR mRNA electroporated cells had comparable IFNγ expression in both PAs and D) MFI decreased in a time-dependent manner by 72 hours after transfection.

Cryopreservation of Electroporated T Cells

Figure 5: MaxCyte electroporation has minimal impact on TCR expression, cell viability and cytokine function upon cryopreservation.
A) Schematic showing primary activated human T cells electroporated with TCR mRNA using a MaxCyte GTx^® . Electroporated cells were divided into 4 groups. The first group was cultured for 24 hours at 37°C and analyzed by flow cytometry. The remaining three cell groups were cultured at 37°C for 1, 4, and 18 hours after transfection, and then cryopreserved. After thawing, these three cell groups were cultured for 21, 18, and 4 hours, respectively, and then analyzed at the same time. B) TCR expression was comparable between fresh and frozen electroporated cells (88-89%). C) Cell viability of the frozen cells (75-76%) was not significantly impacted compared with freshly electroporated cells (82%). D-E) TCR expressing lymphocytes were co-cultured with antigen peptide-pulsed T2 cells and analyzed by flow cytometry. IFNγ expression and MFI were comparable between fresh and frozen electroporated T cells.

MaxCyte Flow Electroporation^® Technology provides:

• High transfection efficiency >90% mRNA TCR expression
• High cell viability >80%
• Scalability from 1.5 x 10⁷ cells in static PAs to 3.9 x 10⁹ cells in a flow electroporation PA
• Minimal impact on cryopreservation of T cells for high TCR expression, high viability, and functionality comparable to freshly electroporated T cells.