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 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.

No significant difference in the survival of metastatic osteosarcoma implanted mice 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. CD80 and CD86 expression on metastatic osteosarcoma was significantly increased after α-PD-L1 mAb treatment. PD-1 expression on CD8+ TILs was significantly decreased after α-PD-L1 mAb treatment suggesting that tumor cells are no longer co-opting the use of PD-1 to suppress T cell function. 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.

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 and 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. This was in comparison to 0% long-term survival of mice treated with α-PD-L1 alone.

CTLA-4/α-PD-L1 mAb treated mice that had controlled initial K7M2 tumors, were completely immune to challenge with additional K7M2 cells, and remained tumor free for an additional 80 days when they were euthanized to evaluate immune memory. The vast majority (86.63 ± 6.661%) of the CD8 cells present in the lungs of combination treated mice were able to produce both IFNy and TNF in response to stimulation with parental K7M2 cells in vitro. All mice that were CD8 depleted prior to K7M2 re-challenge succumbed to metastatic osteosarcoma, with a median survival of ~40 days. This was in comparison to control α-CTLA-4/α-PD-L1 mAb treated tumor-immune mice that were re-challenged with K7M2 cells without CD8 depletion and showed no evidence of lung metastases or disease

No additional improvement in the survival of mice treated with doxorubicin + α-PD-L1 mAb compared to α-PD-L1 mAb treatment alone. combination of chemotherapy with immunotherapy approaches do not appear to have additional beneficial effects on tumor control and mice eventually succumb to disease similar to progression during α-PD-L1 mAb treatment alone.

Higher doses of this CTLA4 mAb clone may be needed to elicit significant survival benefits in CTLA4 mAb treated mice alone, however, at this low dose we were still able to see a synergistic effect when coupled with PD-L1 mAb treatment. This may be of some advantage when treating pediatric patients, as high dose CTLA4 mAb treatment can lead to significant secondary side effects. Thus, combinational α-PD-L1/α-CTLA-4 blockade is an attractive combinational therapy to use in humans.

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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