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Updated: May 7, 2026
A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants
Published on: August 9, 2019
Selective expansion of T-cell receptor engineered T cells with increased stem-like phenotypes using neoantigen
Sanghyun P Kim1, Noam Levin1, Youngseo Jo2
1Surgery Branch, National Cancer Institute, Bethesda, Maryland, USA sar@nih.gov noam.levin@nih.gov peter.kim@nih.gov.
Background:
Adoptive transfer of T-cell receptor-engineered T cells (TCR-T cells) has shown promising efficacy in solid tumor treatment, but achieving clinical benefit typically requires infusion of tens of billions of cells. The commonly used rapid expansion protocol (REP), based on the CD3-agonistic OKT3 antibody and irradiated allogeneic feeder cells, exponentially expands tumor-infiltrating lymphocytes (TILs). However, the effect of REP on TCR-T cell frequency and phenotype remains unclear. This study aimed to evaluate the impact of REP on TCR-T cells and to assess the potential of a neoantigen-specific stimulation platform, NeoExpand, as a strategy to selectively expand TCR-T cells with favorable phenotypes.
Methods:
We compared the effects of REP and NeoExpand on the frequency, yield, and phenotype of TCR-T cells engineered against shared neoantigens. Various autologous antigen-presenting cell (APC) types, including dendritic cells and bulk peripheral blood mononuclear cells (PBMCs), were tested as stimulators in NeoExpand. Additionally, we combined NeoExpand with CD3 activation, with or without allogeneic feeders, to improve scalability. The role of exogenous interleukin (IL)-21 in shaping TCR-T cell phenotypes was also investigated.
Results:
REP impaired the frequency and phenotype of TCR-T cells, particularly CD4+ and CD8+ subsets matched to their engineered TCRs. In contrast, NeoExpand selectively increased TCR-T cell frequency and improved their phenotypes but did not consistently achieve higher total yields compared with REP. Among the tested autologous APCs, PBMCs effectively supported selective expansion. Further optimization using PBMCs as APCs revealed that combining NeoExpand with CD3 activation enabled exponential expansion without compromising selectivity. Inclusion of IL-21 during NeoExpand promoted a naïve/stem-like phenotype in CD8+ TCR-T cells, with minimal effect on CD4+ TCR-T cells.
Conclusions:
These findings demonstrate that NeoExpand enables selective expansion of TCR-T cells while preserving favorable phenotypes and scalability. The approach supports further clinical evaluation of NeoExpand as a strategy to generate high-quality TCR-T cell products for adoptive cell therapy.
