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Background Adoptive transfer of T-cell receptor-engineered T cells (TCR-T cells) has shown promising clinical efficacy in treating solid tumors. Clinical responses in solid tumors may require transfer of tens of billions of T cells. A common method to achieve large-scale TCR-T cell expansion is a rapid expansion protocol (REP) via the anti-CD3 agonistic antibody OKT3 and irradiated allogeneic feeder cells. While a REP can exponentially expand tumor-infiltrating lymphocytes (TILs), its impact on TCR-T cell frequency and phenotype remains unclear.Methods and Results We first examined the effect of REP on the frequency and phenotype of TCR-T cells, taking into account CD4 or CD8 co-receptor expression since some TCRs may require co-receptor expression matched to the engineered TCRs for full activity. TCR-T cells expressing the appropriate co-receptor are hereafter referred to as mTCR +Co-R+ cells. Analysis of TCR-T infusion products as part of clinical trial (NCT03412877) revealed that REP negatively affected the frequency of mTCR+Co-R+ cells (fold reduction: 0.76, p=0.043, N=14). Their phenotype was also adversely affected as REP resulted in increases in CD39 expression and effector memory cells (CD45RO+CD62L-) at the expense of less-differentiated central memory (CD45RO+CD62L+) and naïve/stem-like (CD45RO-CD62L+) cells. To address this challenge and selectively expand mTCR+Co-R+ cells, we applied our previously described NeoExpand protocol, utilizing neoantigen-specific stimulation by co-culturing TCR-T cells with APCs either pulsed with neoantigen peptides or transfected with neoantigen RNAs.When compared to REP, NeoExpand increased mTCR+Co-R+ cell frequency (fold difference: 3.12, p<0.001, N=33) and improved their phenotype by increasing CD39- cells and naïve/stem-like cells. However, it did not consistently generate higher total TCR-T cell numbers than REP, likely due to robust T-cell expansion at the bulk level under REP. Various autologous antigen-presenting cells (APCs) were tested to further improve NeoExpand: bulk peripheral blood mononuclear cells (PBMCs) expanded TCR-T cells as efficiently as dendritic cells (DCs).Surprisingly, combining NeoExpand with REP enabled exponential TCR-T cell expansion without compromising selectivity. Furthermore, the addition of OKT3 alone without allogeneic feeders to the NeoExpand cultures using autologous PBMCs significantly increased TCR-T-cell expansion, suggesting PBMCs can serve as both APCs and feeder cells. Mechanistically, we show that exogenous IL-21 included during NeoExpand was responsible for promoting a naïve/stem-like phenotype in CD8+ TCR-T cells in NeoExpand cultures, with minimal effect on CD4+ TCR-T cells.Conclusions Taken together, these findings support the combination of NeoExpand and CD3 activation as a promising approach for robust, selective TCR-T cell expansion for clinical applications.Ethics Approval This study was approved by the Investigational Review Board at the National Cancer Institute (NCI) in accordance with an assurance filed with and approved by the U.S. Department of Health and Human Services and was registered at https://clinicaltrials.gov under NCT00068003 and NCT03412877.