Background cTRLs are a promising alternative to TILs, chimeric antigen receptor (CAR) and T cell receptor (TCR) T cell therapies for the treatment of solid tumors. cTRLs address several key challenges of TILs, such as the requirement for a surgical procedure and having low numbers of exhausted CD8+ T cells. This study evaluates the therapeutic potential of cTRLs in CRC patients by demonstrating their reactivity against autologous tumors as well as their more favorable phenotypic characteristics compared to TILs.

Methods We have previously described the ability to isolate cTRL using a proprietary magnetic isolation instrument (IsoQoreTM) and the tissue-resident associated surface marker CD103.¹ Using flow cytometry, we compared the phenotypic characteristics of paired cTRLs and TILs from CRC patients by measuring the frequency and expression levels of markers associated with exhaustion (TOX, PD1 and CD39), memory phenotype (CD27 and CD28) and tissue homing receptors (CXCR3, CCR5). Additionally, we isolated cTRLs from colorectal cancer patients (n=6), and assessed their reactivity against autologous tumors in a co-culture assay by measuring the upregulation of intracellular interferon-gamma (IFNγ).

Results TILs exhibited the anticipated exhaustion phenotype characterized by high levels of TOX, PD-1 and CD39. In contrast, cTRLs showed lower levels of exhaustion markers while displaying higher expression of the memory associated markers CD27 and CD28. Notably, cTRLs showed a different expression profile of tissue homing receptors compared to TILs, with higher levels of CXCR3 but lower levels of CCR5, suggesting that these cell types may have distinct homing characteristics. Studies measuring cTRL (CD8+CD103+) tumor reactivity demonstrated robust activity against autologous tumors as measured by an increase in intracellular IFNy production. Importantly, no increased reactivity was observed in non-cTRLs (CD8+CD103-). Collectively, these findings indicate that cTRLs respond to tumors robustly, while possessing a distinct phenotype from TILs and a potentially functional advantage over TILs.

Conclusions These findings highlight the therapeutic potential of cTRLs in colorectal cancer. Sourcing cTRLs from blood, instead of requiring invasive surgery for isolating TILs, not only addresses the challenge of tumor resectability, but also significantly expands the pool of eligible patients. In addition to their specific tumor reactivity, cTRLs also have a more favorable phenotype compared to TILs, including lower expression of exhaustion markers and higher expression of memory markers. These superior characteristics underscore the potential of cTRLs and provide a compelling rationale for further investigation and development of cTRL-based therapies.

Acknowledgements The authors wish to acknowledge Philip Vorhies and Mallory Sievers for logistic support, as well as Discovery Life Sciences and Accio Biobank for sample procurement. We would also like to thank the patients with colorectal cancer who provided samples to inform this work.

Reference

1. Wang Z, Ahmed S, Labib M, Wang H, Wu L, Bavaghar-Zaeim F, Shokri N, Blanco S, Karim S, Czarnecka-Kujawa K, Sargent EH, McGray AJR, Perrot M, Kelley SO. Isolation of tumour-reactive lymphocytes from peripheral blood via microfluidic immunomagnetic cell sorting. Nat Biomed Eng. 2023;9:1188–1203.

Ethics Approval Signed written informed consent is obtained for every study participant prior to their participation. The Informed Consent Form (ICF) receives Research Ethics Board (REB) approval prior to use and continued approval will be granted for any changes that are made to the approved ICF. Before any study procedures are performed, participants will have the details of the study described to them and will be given a written ICF to read. Any pertinent questions regarding the study will be answered by the study personnel. If participants consent to participate in the study, they will indicate consent by signing and dating the ICF in the presence of study personnel.