Evaluating Gene Expression and TCR Repertoire Dynamics in γδ T Cell Therapies

 

Abstract

γδ T cell-based therapies have been advancing clinically and have shown therapeutic promise, but their molecular profiles during manufacturing have not been well characterized. Deeper analysis of gene expression changes through manufacturing may identify predictors of γδ T cell expansion and/or correlate to potency or clinical outcomes. Further, such data may elucidate which profiles or cellular characteristics are imparted by donor selection versus those from the manufacturing process.

We evaluated TCR repertoire and gene expression from both (a) the apheresis starting material and (b) the final manufactured γδ T cell product from healthy donors and glioblastoma (GBM) patients (NCT 04165941). Gamma-delta T cell clinical products were manufactured from apheresis using proprietary expansion protocols. The cells were transduced with a Methylguanine-DNA methyltransferase (MGMT) construct, which conveys resistance to temozolomide chemotherapy. TCR libraries were prepared with VDJ-specific primers for α, β, δ, and γ chains using iRepertoire’s RepSeq+ technology. In parallel, bulk RNA phenotyping libraries were prepared by dimer avoided multiplex PCR (dam-PCR) encoding +150 immune profiling genes and sequenced using Illumina’s platform. Multigroup comparisons were performed using a nonparametric statistical test while controlling for false discovery rates.

At manufacturing initiation, the TCR repertoire consisted primarily of αβTCR clones. Gene expression levels from healthy donors seem to have increased BCL6, naïve cells, CD8, and CD63, but overall gene expression was similar between healthy donors and GBM patients. The corresponding shift to γδ TCR expression was confirmed in the final products, where the preferential expansion of Vγ9 and Vδ2 clones was observed from both healthy donors and GBM patients. Products manufactured from healthy volunteers and GBM patients exhibited highly similar gene expression profiles. Interestingly, gene expression analysis showed a significant increase in markers of cellular activation and cytotoxicity in both final products. These markers indicate that the cells are active and primed to kill. Additionally, the expression of immune trafficking and stimulation markers significantly increased in both final products, suggesting that manufactured cells are capable of tissue trafficking and immune cell recruitment.

Our study shows that activated, cytotoxic γδ T cell products were successfully manufactured from different starting materials and exhibited similar gene expression profiles, which suggests the process is robust and reproducible across different donor populations. In addition, immunophenotyping gene expression of DeltEx DRI product elucidated additional attributes such as cytotoxic and/or trafficking potential and the ability to stimulate immune cell recruitment.

Highlights

• RepSeq+ TCR 4-chain RNA analysis can track shifts in relative amounts of mapped uCDR3 before, during, and after the manufacturing process ​ ​​​​ ​
• Combined immune repertoire and ImmunoSight immunophenotyping analysis indicates that ​the expanded and activated γδ T cells significantly shifted to an increase of γV9 and δV2 clones and expressed activation, cytotoxicity, and cell migration genes

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