451 CD40L-4–1BB can stimulate tumor infiltrating lymphocyte <i>ex vivo</i> expansion from melanoma core biopsies

<h3>Background</h3> Tumor-infiltrating lymphocyte (TIL) expansion <i>ex vivo</i> 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 <i>ex vivo</i> TIL expansion from melanoma core needle biopsies (CNB). <h3>Methods</h3> 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. <h3>Results</h3> 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 &gt;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<i> vs</i> 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⁺<i> vs</i> CD4⁺ T cells (p&lt;0.001). In an HLA-A2 positive sample, co-culture with HLA-matched cell lines resulted in IFNγ release &gt;200pg/ml with &gt;70% HLA blockable, confirming tumor-reactivity. <h3>Conclusions</h3> <i>Ex vivo</i> 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.