Abstract C030: Timing of melanoma brain metastasis seeding dictates the immunomodulatory effect and efficacy of systemic anti-CD40 agonist therapy
Authors: Cheng Vinton W. T. , Breza Victoria R. , Chertok Beata , Sheybani Natasha D. , Bullock Timothy N. J. , Price Richard J. 
Published: November 2024

Abstract
Introduction: Melanoma brain metastasis (MBM) affects nearly 50% of patients with advanced melanoma. Despite progress in systemic therapies, particularly immune checkpoint inhibitors, intracranial responses remain highly variable. The CD40/CD40L axis and its pleiotropic effects on the anti-tumoral immune response has the potential to improve MBM treatment. Our aim was to investigate whether the timing of MBM seeding, in a two-site mouse model of MBM, impacts the intracranial efficacy of anti-CD40 agonism (aCD40). Methods: 2 x 105 B16F1-cOVA murine melanoma cells were subcutaneously (s.c.) injected in the right flank in C57BL/6 mice (5F, 5M). After 3 days, 1 x 105 B16F1-cOVA cells were intracerebrally (i.c.) injected into the right striatum under stereotaxic guidance (early MBM’ cohort). A separate cohort of C57BL/6 mice (5F, 5M) had s.c. injection of B16F1-cOVA cells, followed by an i.c. injection of B16F1-cOVA cells after 5 days (‘late MBM’ cohort). In both experimental cohorts, the mice were randomly allocated to receive either an aCD40 antibody or an isotype IgG antibody (control) intraperitoneally on days 4 (D4) and 7 (D7) post-i.c. injection (D0). S.c. tumor volume and survival data were serially collected. Separately, tumor-bearing mice treated with either aCD40 or IgG (n = 4 per early/late MBM groups) were euthanized day 9 (D9) post-i.c. injection and tissues (blood, brain tumor [BT], and flank tumor [FT]) were collected for immune profiling by spectral flow cytometry. Results: The early MBM cohort showed a significant s.c. tumor response to aCD40 therapy with a mean D9:D4 s.c. tumor volume ratio of 2.81 (SD±2.05) vs 16.51 (±13.10) in the IgG group (unpaired t-test; p=0.0497). The late MBM cohort showed no significant difference in s.c. tumor response between aCD40 or IgG (D9:D4 ratio 1.52±1.00 vs 1.20±0.83; p=0.5999). Survival increased in early MBM following aCD40 vs IgG (median 20 days vs 13 days; logrank test, p=0.0078), which was not replicated in late MBM (median 21 days vs 19 days; p=0.7717). Systemic aCD40 led to reduced circulating B cells, most expressing CD11b, and increased monocytes, compared to IgG treatment. In the BT, there were higher infiltration of CD8 T cells (normalized to microglia), including OVA+ CD8 T cells, and CD4 T cells in early MBM treated with aCD40 vs IgG. Systemic aCD40 induced more infiltrating SIRP1a+ cDC2 cells than XCR1+ cDC1 cells and vice versa in the IgG group, in both the BT and FLT. Meanwhile, infiltrating B cells were predominantly CD11b+ in aCD40 groups in the BT, regardless of MBM timing. Conclusion: Systemic aCD40 therapy improved survival of MBM-bearing mice, with a shorter interval between s.c. and i.c. implantation; an effect abrogated by later MBM seeding. Systemic aCD40 therapy induced specific changes in the BT infiltrative immune profile, which may explain the distinct response between the early and late BM cohorts. These findings have implications on the experimental modelling of MBM and provide insight into variability of MBM response to immunomodulating therapies.
Citation Format: Vinton W. T. Cheng, Victoria R. Breza, Beata Chertok, Natasha D. Sheybani, Timothy N. J. Bullock, Richard J. Price. Timing of melanoma brain metastasis seeding dictates the immunomodulatory effect and efficacy of systemic anti-CD40 agonist therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C030.