Introduction: We have previously shown the successful engineering of membrane-bound IL15 (mbIL15)-expressing tumor-infiltrating lymphocytes (TIL; cytoTIL15 cells) from solid tumors such as melanoma, which are CD8+ enriched TIL exhibiting enhanced persistence and anti-tumor activity compared with unengineered TIL (SITC 2022, 2023; clinical candidate OBX-115: NCT05470283). The mbIL15 expressed by cytoTIL15 cells is regulated via a carbonic anhydrase 2 (CA2) drug-responsive domain, which allows expression to be induced upon delivery of the FDA-approved small molecule ligand acetazolamide (ACZ) and was designed to engage the IL2Rβ and IL2Rγ receptors in both cis and in trans. Using natural killer (NK) cells, which are potently responsive to soluble, trans-presented IL15, as an experimental model system, we examined the potential of mbIL15 on cytoTIL15 cells to potentiate activity in both cis and trans.

Methods: cytoTIL15 cells and unengineered TIL were generated as described previously (SITC 2022, 2023). Cells were assessed for expression of mbIL15 and phosphorylation of downstream signaling molecules STAT5 and S6 by flow cytometry, and co-cultured with allogeneic healthy donor NK cells for 1-10 days with and without transwell inserts. Functional effects of cytoTIL15 cells were compared to those of soluble recombinant IL15. Supernatant was obtained from co-cultures for assessment of cytokine production.

Results: cytoTIL15 cells demonstrated robust ACZ-dependent mbIL15 expression, and ACZ dosing regulated mbIL15 activity in cis by potentiating the phosphorylation of STAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.012) and S6 (geoMFI with 0 vs. 25 µM ACZ, p=0.016). Upon removal of ACZ, mbIL15 expression and pSTAT5 and pS6 returned to basal levels within 24 hours. mbIL15 expressed on the surface of cytoTIL15 cells could also activate cells in trans in an ACZ-dependent titratable manner. Specifically, co-culture of cytoTIL15 cells with NK cells led to an ACZ-dependent increase in pSTAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.038) in NK cells, which was not seen in co-cultures of unengineered TIL with NK cells. Additionally, NK cells showed a similar cytokine production profile when exposed to mbIL15 by cytoTIL15 cells as with soluble, trans-presented IL15. NK cell transactivation relied on direct cell contact with cytoTIL15 cells, as magnitudes of phosphorylation were decreased in co-cultures with NK and cytoTIL15 cells separated by a transwell membrane (pSTAT5 geoMFI p=0.008).

Conclusions: cytoTIL15 cells demonstrate functional engagement of cytokine receptors via mbIL15 in both cis and in trans, including phosphorylation of STAT5 and S6. Taken together, these data highlight the potential for robust activity of cytoTIL15 cells through potentiation of ACZ-dependent mbIL15 activity in both cis and trans.

Citation Format: Rachel Burga, Gauri Kulkarni, Zheng Ao, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB072.