Background
Tumor-infiltrating lymphocyte (TIL) expansion ex vivo is currently performed from surgically excised tumors in culture media containing Interleukin-2 (IL-2). Targeting antigen-presenting cells through CD40 and tumor-reactive T cells through 4-1BB signaling may result in more robust anti-tumor activity. In this study we investigated the impact of coordinated CD40L and 4-1BB costimulation on ex vivo TIL expansion from melanoma core needle biopsies (CNB).
Methods
As part of an IRB approved protocol, additional CNB passes were obtained from melanoma patients utilizing ultrasound- or CT-guidance with 18- or 20-gauge needles as part of their clinical care. Each core was plated individually in a G-Rex 24 well plate in 8 mL of culture media containing IL-2 (6000 IU/mL) and bispecific CD40L/4-1BBL fusion protein (43nM) with additional IL-2 on days 7, 14, 21. Resulting TIL were harvested on day 28. Flow cytometry was performed on TIL and tumor-reactivity was measured with HLA-matched melanoma cell lines. Tumor immune microenvironment (TIME) characteristics of core biopsy tissue were correlated with expanded TIL count.
Results
We enrolled 17 melanoma patients, 7 female (41%) and 10 male (59%), median age= 72 [IQR, 68–78], 10 (59%) treatment-naïve, and 7 (41%) on-treatment (immune checkpoint blockade n=6 and targeted therapy n=1). CNB were obtained from lymph node (n=7, 41%), liver (n=3, 18%), lung (n=3, 18%), subcutaneous and soft tissue [SSTs] (n=4, 24%). TIL was successfully expanded (≥10e6) from CNB in 15 (88%) of the cases. Among successfully expanded cases, 2 (13.3%), 3 (20%), and 10 (66.7%) grew 10-20e6, 20-50e6, and >50e6 TIL, respectively. Median TIL count varied based on tissue of origin and was 399e6, 174e6, and 42e6 from lymph nodes, visceral organs, and SSTs, respectively. TIL growth kinetics varied among cores from the same patient. There was a trend towards higher TIL count expanded from treatment-naïve vs those patients on treatment (293e6 vs 27e6, respectively, p=0.05). There was no correlation between number of cores and expanded TIL count (r=0.19), however, the degree of TIL infiltration in tumor tissue was associated with higher expanded TIL count (p=0.02). Flow cytometric analysis of TIL revealed a significantly higher proportion of CD8+ vs CD4+ T cells (p200pg/ml with >70% HLA blockable, confirming tumor-reactivity.
Conclusions
Ex vivo CD40L-4-1BBL costimulation can result in successful expansion of CD8+ predominant TIL from melanoma CNB. Further data on TIL phenotype in association with TIME will be presented as well.