Combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma

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Combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma(1)

In the K7M2, PD-L1 knock-down cells, mouse model of metastatic osteosarcoma, expression of programmed death receptor-1 (PD-1) and interaction with its ligand PD-L1 on tumor cells, tolerizes tumor-reactive T cells inhibiting their cytokine production and cytotoxic function towards the tumor. These immune checkpoint blockade strategies also appear to enhance clearance of tumors when used in combination with chemotherapy, including during treatment of pancreatic cancer in murine models.

No significant difference in the survival of metastatic osteosarcoma implanted mice was obtained when treated for 30 days vs 15 days; mice receiving α-PD-L1 mAb therapy perished from pulmonary metastases with a median survival of 68 days in both groups. PD-L1 expression on metastatic osteosarcoma was significantly decreased after α-PD-L1 mAb treatment suggesting that these cells are no longer co-opting the use of PD-1 to suppress T cell function. Moreover, CD80 and CD86 expression on metastatic osteosarcoma was significantly increased after α-PD-L1 mAb treatment suggesting that tumor cells may be directly or indirectly using this pathway to suppress CTL-mediated killing. PD-1 expression on CD8+ TILs was significantly decreased after α-PD-L1 mAb treatment. Tumor infiltrating CD8 T cells had higher levels of CTLA-4 expression in α-PD-L1 mAb blockade treated mice. No statistical difference was observed in LAG3 expression after α-PD-L1 mAb treatment.

Lower expression of PD-L1 on K7M2 metastatic osteosarcoma cells from α-PD-L1 mAb-treated mice compared to tumor cells from mock-treated mice. Metastastatic osteosarcoma cells isolated from α-PD-L1 mAb treated mice were re-implanted into naïve recipient mice that were then subsequently treated with α-PD-L1 mAb or received mock injections. mice injected with osteosarcoma cells from α-PD-L1 mAb treated mice and treated with additional α-PD-L1 mAb showed no difference in survival compared to mock treated mice. Tumor cells from α-PD-L1 mAb treated mice retained low expression levels of PD-L1, compared to tumor cells from untreated mice, whether or not additional α-PD-L1 mAb was administered. CD8+ TILs isolated from PD-L1 mAb resistant tumors continued to exhibit decreased expression of PD-1 but elevated expression of CTLA-4, while LAG3 expression remained unchanged.

Combination of α-CTLA-4 and α-PD-L1 mAb treatment resulted in complete control of metastatic osteosarcoma tumors with long-term disease-free survival in roughly 60% of α-CTLA-4 + α-PD-L1 mAb treated mice.

Mice receiving immunotherapy with α-PD-L1 mAb eventually succumb to pulmonary metastatic disease. Here we show that metastatic osteosarcoma tumors from mice treated with α-PD-L1 mAb down-regulate expression of PD-L1, and become resistant to further blockade treatment.

Combinational α-PD-L1/α-CTLA-4 blockade is an attractive combinational therapy to use in humans to enhance T cell mediated rejection of metastatic osteosarcoma with the ultimate goal of improving patient prognoses while avoiding tumor escape. Alternative immune checkpoint controls (CTLA-4, PD-1, LAG-3, TIM-3) on T cells provide tumors with several options for avoiding tumor-reactive immune responses.

1. D. M. Lussier, J. L. Johnson, P. Hingorani, J. N. Blattman, Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma. J Immunother Cancer. 3, 21 (2015).

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