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Background:

• The Immune Synapse is a membrane complex of co-stimulatory molecules (co-stim), antigens, and other factors organized into a higher order structure.
• Current cell expansion technologies can’t replicate this organization or mediate selective cell expansion.
• Programming molecular mediators of immune activity is necessary to selectively expand specific immune phenotypes.
• Red Blood Cells (RBCs) provide a biocompatible framework to present costim molecules in a controlled manner.

Rationale:

• To advance CAGT innovation and clinical impact, we developed a customizable Programmable Cellular Interface (sbPCI) on an RBC platform with proof of concept demonstrated initially on sbPCI-1.

Methods:

• Autologous and allogeneic RBCs were modified to display an array of sbPCI-1 molecular configurations.
• PBMCs from 3 donors were co-cultured at a 1:10 ratio (sbPCI-1:PBMC ) and exogenous IL-2 (50U/ml) & IL-21 (30ng/ml) added 3x per week.
• Lymphocytes were re-stimulated and analyzed every 8 days.
• Total RNA was extracted from all 3 donor PBMCs, plus corresponding expanded T cells at Days 8 and 24.
• cDNA was synthesized for qPCR use with TaqMan gene expression assays.
• Error bars are Standard Error of the Mean (SEM).

Figure 1: Programmable Cellular Interface Platform. RBCs were isolated from donor blood and modified to display sbPCI consisting of co-stim molecules. Lymphocytes were then expanded on sbPCI.

Results:

Figure 2: Performance Comparison: sbPCI-1 vs. Leading Competitors. PBMCs were expanded, according to manufacturer’s directions, and compared with sbPCI-1. The sbPCI-1 did not require the initial T-cell isolation step. A) sbPCI-1 had improved fold expansion over 16 days. B) sbPCI-1 showed similar performance, with a decrease in CD4:CD8 ratios observed over a period of 16 days. P-values calculated using a two-way ANOVA (*p < 0.05, **p < 0.01) suggested differences observed were significant. These observations supported further development of the sbPCI system.

Figure 3: Modular Design for Rapid Modification of the Molecular Formats. A) Molecular configurations can be easily optimized with the sbPCI system to drive desired outcomes. B) Unstimulated PBMCs co-cultured 48 hours with sbPCI, harvested and then analyzed for Granzyme B expression by flow cytometry. C) Cells were enumerated over 24 days to determine total cell expansion. D) 24-day fold expansion was calculated based on cell numbers with sbPCI-1 iii showing a reduced fold expansion by Day 24.

Figure 4: Molecular Formats Selectively Expand Central Memory (CM) T-cells vs Effector Memory (EM) T-cells. PBMCs were co-cultured with sbPCI over 24 days and analyzed by flow cytometry and gene expression every 8 days. A) All sbPCI formats produced primarily CM T-cells by Day 8 and maintained this phenotype. B) At Day 8, the majority of T cells were positive for CCR7+. By Day 24, there was an increased number of CD3+CCR7+ expressing cells in the sbPCI-1 iii cultures compared with other sbPCI-1 configurations.

Figure 5: Molecular Formats Increase CD3+CD8+. A) Day 24 expanded cells’ phenotype was evaluated by flow cytometry and expression measured as the percent positive. B) The sbPC-1 iii expanded a majority of CD3+CD8+.

Figure 6: T-cell Activation Pathways Are Upregulated on Day 24. mRNA was analyzed for log2 fold changes between formats, with unpaired t-tests determining significance (*p < 0.05). CTLA-4 expression was dependent on the sbPCI-1 format.

Conclusion:

• The molecular configuration of co-stim molecules presented to PBMCs can be easily optimized using the sbPCI system.
• Changes in molecular formats affect T-cell expansion and phenotype.
• Expansion ranged from 1,115x to 2,025x, without the need for T-cell isolation.
• sbPCI-1 iii expanded primarily CD8+ Tcells, suggesting the CD3+CD8+ isolation step could be removed.
• sbPCI-1 supported CM T-cells expansion.
• CTLA-4 expression was influenced by the sbPCI-1 molecular format.

Future Directions:

• These findings warrant further experiments testing types, placement, and concentrations of different co-stim molecules and antigen combinations.
• Future sbPCI molecular formats aim to enhance cell therapy precision by focusing on: CAR-T, TCR-specific, NK/NKT cells, and iPSCs.
• Collaborations with manufacturing sites to demonstrate patient-specific use.