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366 Neoantigen-specific stimulation of T cells for effective cancer adoptive cell therapies
  1. Sanghyun Kim1,
  2. Noam Levin2,
  3. Charles Marquardt1 and
  4. Steven A Rosenberg1
  1. 1National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
  2. 2NCI, Rockville, MD, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Targeting neoantigens by adoptive cell therapy (ACT) can effectively treat advanced solid tumors.1–5 However, the conventional rapid expansion protocol (REP) for T-cell expansion can stimulate bystander cells6–8 and cause differentiation and exhaustion of T cells.9–11 This can lead to inadequate expansion of neoantigen-reactive T cells and hence ineffective ACT.

Methods We developed an in vitro culture method, termed NeoExpand, where T-cell receptor-engineered T cells (TCR-T) or neoantigen-reactive tumor infiltrating lymphocytes (neoTIL) were selectively expanded by neoantigen-specific stimulation. Briefly, T cells were co-cultured with antigen presenting cells or engineered cell lines loaded with neoantigens for ~2 weeks in the presence of interleukins 2 and 21.

Results When NeoExpand was used to expand TCR-T cells expressing previously identified CD8+ TCRs targeting shared p53 or KRAS neoantigens,4 12 selective expansion of TCR-expressing CD8+ T cells were observed when compared to REP [1.6 fold, p<0.001, n=8 (TCRs)]. Phenotypically, NeoExpand expanded CD39-CD69- cells, reportedly less differentiated T cells with stem-like features,7 relative to REP (9.9 fold, p<0.001, n=12).

Next NeoExpand’s ability to facilitate neoantigen-reactive TCR isolation was tested. From 25 TIL samples from tumors expressing p53 or KRAS mutations, the conventional screening13 identified 14 neoTIL clonotypes (i.e., neoantigen-reactive TCRs) (3 CD4; 11 CD8), while NeoExpand enabled identification of 42 clonotypes (14 CD4; 28 CD8), indicating neoTIL’s repertoire expansion during NeoExpand.

Next, we examined the effect of NeoExpand on expansion, phenotypes and functions of neoTIL. When 11 TIL samples from patients with p53-mutated or RAS-mutated gastrointestinal or breast cancer were tested, greater expansion of neoTIL with NeoExpand was noted relative to REP (4.0 fold, p=0.02). Single-cell transcriptome analysis revealed expansion of neoTILs with stem-like memory cell phenotypes uniquely in the NeoExpand conditions. These neoTILs expressed stem and memory markers, including CD62L, IL7R, and TCF1 and lacked exhaustion-associated gene expression, including CD39 and TIM3. Finally, TILs expanded through NeoExpand or REP were functionally compared using xenograft mouse models. Three TIL samples, one containing p53R175H-reactive TILs and two containing KRASG12V-reactive TILs were expanded through NeoExpand or REP and were adoptively transferred to NSG mice engrafted with p53R175H+ TYK-nu human ovarian cancer cells or KRASG12V+ patient-derived xenograft cancer cells. TILs expanded through NeoExpand led to significant tumor regression (p<0.001, n=5 mice/group).

Conclusions Collectively, NeoExpand selectively expands neoantigen-reactive T cells compared to REP and enables sensitive identification of neoantigen-reactive TCRs by expanding neoTIL repertoire. NeoExpand’s ability to enhance phenotypes and functions of neoantigen-reactive T cells warrants its evaluation for clinical use.


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