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

Abstract

BackgroundWhile immune checkpoint blockade is regarded as standard of care for treatment of non-small cell lung cancer (NSCLC), up to 50% of patients with metastatic NSCLC do not achieve an optimal response.1–3 Previous work by our group and others in adoptive cell therapy (ACT) of metastatic melanoma (MM) has shown that infusion of a CD8+-rich TIL product significantly improved clinical outcomes, yet traditional IL-2 expansion methods have resulted in a predominantly CD4+ NSCLC TIL expansion product.7–12 This preclinical study explores the feasibility of producing a tumor-specific, CD8+-enriched NSCLC TIL product for ACT with an improved culture method.MethodsTIL from resected NSCLC tumors were cultured using 1) the traditional method using 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). Expanded TIL were phenotyped using flow cytometry for CD4 and CD8 subset assessment and the CDR3-beta variable region of the T-cell receptor (TCR) involved in antigen binding was sequenced to assess the T-cell repertoire.ResultsIn a shorter manufacturing time (median of 14 days vs 27.5 days), TIL 3.0 expanded on average 5.3-times more NSCLC TIL (95% CI= 4.3–6.2, p<0.0001) and achieved a higher expansion success rate than the traditional TIL 1.0 method (100% vs 62.5%, respectively, p<0.0001). Additionally, TIL 3.0 greatly enriched for CD3+CD8+ TIL (81.8% vs 36.9%, p=0.001) and expanded a larger breadth of clonotypes (p=0.039) which shared greater homology with the total clonotypes found in the repertoire of the resected tumor (p=0.0007), and contained a greater fraction of the clones found at high frequency in the tumor (p<0.00001). TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared to TIL 1.0 (p=0.0039), defined based on exclusion of known viral-specific TCR and other TCR found in the paired uninvolved lung tissue.ConclusionsThis study reports the feasibility of using the TIL 3.0 methodology to robustly expand a CD8+ T-cell repertoire which maintains the respective clonal hierarchy in NSCLC tumors and enriches for putative tumor-specific TIL clones. The robustness and speed of the new process may facilitate testing and implementing effective TIL ACT in NSCLC.ReferencesGaron EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 2015;372(21):2018–28.Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. 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This study was approved by the University of Texas MD Anderson Cancer Center‘s Institutional Review Board (protocol number PA15-1112_MODCR001).