Tumor reactive T cells from the cerebrospinal fluid of patients with leptomeningeal disease from melanoma.
Authors: Pina Yolanda , Carter Michael , Hall Maclean , Rodriguez Valentin Madeline , Beatty Matt , Forsyth Peter A. J. , Pilon-Thomas Shari 
Published: June 2024

2027

Background: Leptomeningeal disease (LMD) is a devastating complication from cancer, with a median survival of 6-8 weeks. The incidence of LMD is highest in melanoma (5-25%) carrying the worst prognosis. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) showed complete and durable responses in patients with advanced metastatic melanoma. CSF-derived T cell populations in LMD shift to more exhausted phenotypes compared to extracranial and brain metastases. Ex vivo large T cell expansion can overcome this shortfall, with adoptive T cell transfer representing a novel therapeutic approach to treat melanoma LMD (M-LMD). The goal of this study is to optimize rapid CSF-derived tumor reactive T cell expansion in M-LMD, and evaluate the immune microenvironment in CSF compared to peripheral immunity and anti-tumoral response. Methods: CSF was collected from M-LMD patients (n=6) via lumbar puncture or Ommaya. Cells were plated following established TIL culture protocols to determine optimal expansion conditions i.e.: 1) 6000 IU/mL IL-2, 2) IL-2+OKT3, 3) IL-2+anti-4-1BB agonistic antibody, 4) IL-2+IL-7+IL-15+IL-21, 5) IL-2+anti-CD3/CD28 human dynabeads, & 6) IL-2+anti-CD3/CD28/CD137 human dynabeads. After 4 weeks, T cells were propagated via rapid expansion protocol (REP) and phenotyped for CD4+ and CD8+ T cells with flow cytometry. T cells were co-cultured for 18-24 hours with HLA-matched melanoma cell lines to assess functionality, and IFN-y levels evaluated in supernatant. Ongoing studies are evaluating other biomarkers i.e., chemokines via Luminex. To examine the expanded TCR clonotypes, TCR beta seq was done in CSF-derived T cells at different times point of expansion and peripheral T cells. Results: Collected CSF yielded an average 8.12e4 viable cells for expansion (range 5e2-2.43e6). After initial culture in IL-2, 61.9% of samples showed increased cell yield with an average 101.68-fold expansion (range 2.35-1054). PreREP T cells were predominantly CD4+ (45.7%). REP was completed with a 100% success rate (mean 187.47-fold expansion). Post-REP flow cytometry analysis revealed similar results with further expansion of T cells. Additional culture supplements (Methods #2-6) produced similar expansion with a reduced T cell input requirement and demonstrated the potential to enrich for CD8+ T cells. M-LMD T cells were highly functional, producing substantial levels of IFN-y in response to HLA-matched melanoma cell lines. Anti-CD3/CD28 produced a larger expansion of T cells with higher levels of IFN-y. Preliminary TCR beta sequencing data revealed a distinct clonal distribution between peripheral and CSF-derived T cells. Conclusions: Therapies for LMD patients are desperately needed. Results demonstrate successful expansion of T cells ex vivo from CSF in M-LMD. Results raise potential to use autologous CSF-derived T cells as therapeutic strategy for patients with LMD.