Costimulatory effect of agonistic 4-1BB antibody on proliferation and effector phenotype of tumor-infiltrating lymphocytes in melanoma.

Abstract

2511 Background: Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) is a promising form of immunotherapy with a 20-30% durable response rate. Its widespread implementation is limited by the relatively long time needed to establish TIL in vitro and the lack of effective tumor reactivity. Our objective was to incorporate co-stimulation with 4-1BB agonism to optimize TIL growth preparation time and tumor reactivity to improve outcomes. Methods: 7 metastatic melanoma patients were consented to an IRB-approved tissue procurement protocol. For each specimen, TIL were propagated in vitro from 4 melanoma tumor fragments per condition with standard 6000 IU/mL of IL-2 and either 10 ug/mL of 4-1BB agonistic antibody (BMS-663513) or its isotype control. After 3 weeks, TIL were counted and phenotyped using flow cytometry. TIL were re-stimulated with HLA-matched melanomas and assessed for interferon-γ (IFNγ) release by ELISA. Statistical comparisons involved the 2-tailed, paired t test using GraphPad Prism software. Results: 4-1BB treatment yielded a median 4.3x107 TIL (range 1.1x107-8.4x107) compared to a median 2.3x107 cells in the control (range 1.5x106-5.3x107, p = 0.009). This is clinically significant as the initial minimum target number for TIL generation is 3x107. On phenotypic analysis compared to control, 4-1BB treatment resulted in a 1.5X higher CD8+ cell numbers (p=0.02), a 6X lower CD4+ cell numbers (p=0.02), and 5.5X lower CD4+CD25+FoxP3+ Treg cell numbers (p=0.09). A paired sample was re-stimulated in triplicate with HLA-matched tumor target, and ELISA of the supernatant treated with 4-1BB yielded 1.6 fold higher IFNγ (p=0.05). A paired sample was then assayed for known markers of T cell cytolytic activity: EOMES and perforin. 4-1BB treatment resulted in 34.5% EOMES and perforin double positive TIL compared to 18.8% in the control group. Conclusions: 4-1BB agonism is associated with enhanced kinetics of TIL proliferation, increased yield of CD8+ TIL, lower Treg numbers, increased markers of cytolysis, and enhanced IFNγ release upon tumor re-stimulation. 4-1BB agonism may be a useful adjuvant in the generation of TIL in future clinical trials.