Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes

Background Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical study explores the feasibility of producing a NSCLC TIL product with sufficient numbers and enhanced attributes using an improved culture method. Methods TIL from resected NSCLC tumors were initially cultured using (1) the traditional method using interleukin (IL)-2 alone in 24-well plates (TIL 1.0) or (2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). TIL subsequently underwent a rapid expansion protocol (REP) with anti-CD3. Before and after the REP, expanded TIL were phenotyped and the complementarity-determining region 3 beta variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire. Results TIL 3.0 robustly expanded NSCLC TIL while enriching for CD8(+) TIL in a shorter manufacturing time when compared with the traditional TIL 1.0 method, achieving a higher success rate and producing 5.3-fold more TIL per successful expansion. The higher proliferative capacity and CD8 content of TIL 3.0 was also observed after the REP. Both steps of expansion did not terminally differentiate/exhaust the TIL but a lesser differentiated population was observed after the first step. TIL initially expanded with the 3.0 method exhibited higher breadth of clonotypes than TIL 1.0 corresponding to a higher repertoire homology with the original tumor, including a higher proportion of the top 10 most prevalent clones from the tumor. TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared with TIL 1.0. Numerical expansion of TIL in a REP was found to perturb the clonal hierarchy and lessen the proportion of putative tumor-specific TIL from the TIL 3.0 process. Conclusions We report the feasibility of robustly expanding a T-cell repertoire recapitulating the clonal hierarchy of the T cells in the NSCLC tumor, including a large number of putative tumor-specific TIL clones, using the TIL 3.0 methodology. If scaled up and employed as a sole expansion platform, the robustness and speed of TIL 3.0 may facilitate the testing of TIL-ACT approaches in NSCLC.