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

Abstract

BackgroundAdoptive 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.MethodsTIL 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 β variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire.ResultsTIL 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.ConclusionsWe 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.

Infiltration of CD3+ and CD8+ lymphocytes in association with inflammation and survival in pancreatic cancer

“…IL-2 is a cytokine responsible for the growth, proliferation, and survival of T-cells. IL-2 has been shown to play a vital role in the regulation of CD8+ T-cells to maintain their reactivity against tumor cells [ 35 , 36 ]. Our data suggests that IL-1B may play a more dominant role in regulating T-cell recruitment into the tumor microenvironment.…”

Downregulation of hsa_circTLK1 represses non-small cell lung cancer progression by regulating miR-876-3p/SRSF7 axis

“…The immune escape of tumor cells is closely related to T cells, which regulate the body's immune response [ 29 ]. IFN-γ, IL-2 and TNF-α are important cytokines that regulate immune response and play an important role in the body's immune response, which secreted by T cells activation [ [30] , [31] , [32] ]. It was found that the levels of IFN-γ, IL-2 and TNF-α increased significantly after transfection of si-circTLK1 in co-cultured cells.…”

Key oncogenic signaling pathways affecting tumor-infiltrating lymphocytes infiltration in hepatocellular carcinoma: basic principles and recent advances

“…Innovative TIL cultivation methods have emerged to precisely and rapidly obtain tumor-reactive TILs in the laboratory. In the pre-Rapid Expansion Protocol (pre-REP) phase, block-processed tumors are co-cultured with IL-2, CD3 antibodies, and 4-1BB antibodies to activate T cells, enhancing the success rate of TIL cultivation ( 121 , 123 , 124 ). This approach shortens the cultivation time and increases the number of expanded CD8 + TILs ( 123 ).…”

“…In the pre-Rapid Expansion Protocol (pre-REP) phase, block-processed tumors are co-cultured with IL-2, CD3 antibodies, and 4-1BB antibodies to activate T cells, enhancing the success rate of TIL cultivation ( 121 , 123 , 124 ). This approach shortens the cultivation time and increases the number of expanded CD8 + TILs ( 123 ). Generally, TILs producing IFN-γ in co-culture with autologous tumor cells are considered tumor-reactive and are then expanded further, although this requires a longer cultivation time ( 125 , 126 ).…”