RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy [proliferation]

Tumor-infiltrating lymphocytes (TILs) are a promising autologous cell therapy to treat solid tumors. TILs are manufactured by expanding and reinfusing tumor-reactive T cells from tumor biopsies. Efficacy of TIL therapies has been limited by the heterogeneity of expanded TIL products and the high prevalence of dysfunctional exhausted CD8+ T cells (TEX). A subset of CD8+ TILs that are double-positive (DP) for CD103 and CD39 is enriched for tumor-reactive TILs across multiple cancer types, but also is susceptible to the TEX state. We screened overexpression of all human transcription factors (TFs) to discover master regulators of expansion in DP TILs from non-small cell lung cancer patients. RELB emerged as the dominant hit driving proliferation of DP TILs despite exhaustion induced by repeat stimulation, with a skew towards CD8+ cells. TCR-seq showed maintenance of TCR diversity from the initial TCR repertoire after multiple days of in vitro expansion driven by RELB. Transcriptome profiling of multiple RELB-expressing TIL subtypes revealed a shift towards a memory/costimulatory-like phenotype. Using a HER2-targeting CAR and tumor co-culture model, RELB conferred improved persistence after multiple tumor challenges in vitro and improved solid tumor control in mouse xenografts in vivo. Finally, co-culture of RELB-overexpressing TILs with patient-matched tumor organoids showed an increase in TIL product polyfunctionality, tumor reactivity, and tumor killing. Overall design: CD8+ TILs were isolated using a TIL CD8+ magnetic isolation kit (REAlease® CD8 (TIL) MicroBead Kit, human, Miltenyi) and DP TILs were FACS sorted (n = 2 patient samples), activated with Transact (Miltenyi) according to manufacturer's instructions, and transduced (n = 2 transduction replicates) with the MORF library at low MOI. Cells were expanded for 3 days, selected with puromycin at 1 µg/mL for 3 days, and then expanded for an additional 9 days. Cells were then re-activated at day 12 post-transduction with a 3:1 ratio of CD3/CD28 dynabeads (Invitrogen) to T cells, stained with CellTrace Violet (Invitrogen), and sorted for lower and upper 15% tails of CellTrace signal. All replicates were maintained and sorted at a minimum of 400x coverage. Cells were maintained at 1–2 × 106 cells ml-1 for all expansion steps unless otherwise indicated.