Abstract 5109: The anti-PD1+anti-LAG3 and anti-CTLA4+anti-PD1 combinations have non-overlapping mechanisms of action in melanoma cranial and extra cranial metastases
Authors: Phadke Manali S. , Li Jiannong , Mandula Jessica , Chen Ann , Rodriguez Paulo C. , Smalley Keiran S. M. 
Published: April 2023

Abstract
Around 40-60% of patients with advanced melanoma develop brain metastases. Recent clinical studies have demonstrated that the efficacy of anti-PD1+anti-LAG3 may be equivalent to anti-PD1+anti-CTLA4. The goal of our project is to understand the mechanisms of action and resistance to anti-PD1+anti-LAG3 in mouse model of melanoma brain metastases. We used orthotopic syngeneic mouse model in which SM1 and B16100kVII2 cells are injected stereotactically into the brain and into the flank. The mice were treated with anti-PD1+anti-LAG3 and anti-PD1+anti-CTLA4 every 5 days. Single cell RNA-seq platform was used to determine the impact of both the combination therapies upon each individual cell type. Flow cytometry experiments were carried out to assess the percentage of immune cells recruitment. The in vivo studies demonstrated that the anti-PD1+anti-CTLA4 had similar efficacy to anti-PD1+anti-LAG3 at both cranial and extracranial sites over a period of 80 days in the SM1 model. In the less responsive B16100kVII2 model, anti-PD1+anti-LAG3 was initially less effective, but similar numbers of mice failed both each combination. scRNA-seq and IHC analysis of responding tumors showed that the anti-PD1+anti-LAG3 led to a greater accumulation of B cells, DCs, CD4+ T cells and different subsets of CD8+ T cells. Of note, the anti-PD1+anti-CTLA4 was associated with infiltration of Tregs, whereas the anti-PD1+anti-LAG3 was associated with CD4+ Th1 helper cells. Depletion of CD4+ T cells decreased responses to anti-PD1+anti-LAG3 in both the cranial and extracranial tumors, with no change in the magnitude of response to the anti-PD1+ anti-CTLA4. By contrast, depletion of CD8+ T cells suppressed responses to both combinations. Depletion of CD4+ T cells decreased activated CD8+ T cells by 30% in anti-PD1+anti-LAG3 treated tumors and conversely increased numbers of activated CD8+ T cells by 40 % in anti-PD1+anti-CTLA4 tumors. In anti-PD1+ anti-LAG3 treated tumors, T helper function was directed towards CD8+ T cells and not B cells, with B cell depletion impacting responses to both combination immunotherapies in a similar manner. Analysis of relapsing brain tumors from both anti-PD1+anti-LAG3 treated mice showed an increased association of MDSCs and loss of tumor reactive CD8+ T cells. The anti-PD1+anti-LAG3+anti-CTLA4 triplet had a good efficacy and suppressed the growth of both flank and brain tumors over a period of 35 days, an effect associated with increased CD8+ T cell infiltration and reduced numbers of MDSCs and Tregs.We show that anti-PD1+anti-LAG3 can drive CD4+ Th1 T helper cell responses hence increasing the accumulation of effector CD8+ T cells and B cells in melanoma and melanoma brain metastases, improving the durability of response. The triplet combination maximizes the efficacy and duration of response in treatment of melanoma brain metastases.
Citation Format: Manali S. Phadke, Jiannong Li, Jessica Mandula, Ann Chen, Paulo C. Rodriguez, Keiran S. M. Smalley. The anti-PD1+anti-LAG3 and anti-CTLA4+anti-PD1 combinations have non-overlapping mechanisms of action in melanoma cranial and extra cranial metastases. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5109.