Optimizing clear cell renal cell carcinoma tumor-infiltrating lymphocytes under controlled hypoxic conditions.

Abstract

581 Background: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) represents a promising approach for advanced solid tumor treatment. Historically, the success of TIL therapy in clear cell renal cell carcinoma (ccRCC) has been limited by difficulty expanding cytotoxic functional TILs. Hypoxic conditions in the ccRCC tumor microenvironment have been associated with mixed tumor characteristics, including metabolic dysregulation and tumor progression. Given the significant role of hypoxia in ccRCC progression, we investigated TIL expansion under controlled hypoxic conditions and its impact on TIL cytotoxic functions. Methods: Fragments or tumor digests from 41 ccRCC patients were cultured with high-dose (6000IU/mL) IL-2 for four weeks. TIL expansion success was defined as at least one fragment expanding to a minimum of 2 wells. Primary TIL underwent rapid expansion protocol (REP) at 20% (normoxic) or 5% (hypoxic) O 2 levels. Cells were rested in post-REP media for 3–4 days in their respective conditions (20% or 5% O 2 ) and subsequently collected for downstream analysis. Reactivity to autologous tumor, expression of activation/co-inhibitory markers, and memory phenotype were analyzed with flow cytometry and co-culture assays. Results: TILs were successfully grown in 87.8% of samples (36/41). TILs secreted IFNγ in response to autologous tumors in 88.6% (31/35) of the samples. Both TIL expansion and reactivity occurred independently of tumor stage or grade. TIL REP under hypoxic conditions (5% O2) was successful, and these conditions salvaged more CD8+ TILs compared to normoxic conditions (p=0.0382). Hypoxic REP TILs showed increased IFNγ, TNFα, and Granzyme B release in response to autologous tumor compared to normoxic conditions. Additionally, hypoxic REP TILs expressed higher LAG3 and TIM3 markers (p<0.05), with significantly increased proportions of tissue-resident memory-like (TRM) CD8+ T-cells (CD69+CD103+) compared to normoxic conditions (18.2% CD69+CD103+ versus 2.6%, respectively, p<0.0001). These features have been associated with clinical responders TILs in melanoma. Conclusions: This study demonstrates the feasibility of expanding tumor-reactive TILs from ccRCC despite tumors harboring exhausted CD8+ T-cells. Exposing TILs to hypoxic conditions enhanced effector functions and promoted the acquisition of a TRM T-cell phenotype. These findings suggest that ccRCC TIL expansion under controlled hypoxia is feasible and could confer improvement to the clinical efficacy of TIL therapy for ccRCC.