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

Abstract

Background: Osteosarcoma is one of the most common bone cancers in children. Most patients with metastatic osteosarcoma die of pulmonary disease and limited curative therapeutic options exist for such patients. We have previously shown that PD-1 limits the efficacy of CTL to mediate immune control of metastatic osteosarcoma in the K7M2 mouse model of pulmonary metastatic disease and that blockade of PD-1/PD-L1 interactions can partially improve survival outcomes by enhancing the function of osteosarcoma-specific CTL. However, PD-1/PD-L1 blockade-treated mice eventually succumb to disease due to selection of PD-L1 mAb-resistant tumor cells. We investigated the mechanism of tumor cell resistance after blockade, and additional combinational therapies to combat resistance.

Methods: We used an implantable model of metastatic osteosarcoma, and evaluated survival using a Log-rank test. Cellular analysis of the tumor was done post-mortem with flow cytometry staining, and evaluated using a T-test to compare treatment groups.

Results: We show here that T cells infiltrating PD-L1 antibody-resistant tumors upregulate additional inhibitory receptors, notably CTLA-4, which impair their ability to mediate tumor rejection. Based on these results we have tested combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade in the K7M2 mouse model of metastatic osteosarcoma and show that this results in complete control of tumors in a majority of mice as well as immunity to further tumor inoculation.

Conclusions: Thus, combinational immunotherapy approaches to block additional inhibitory pathways in patients with metastatic osteosarcoma may provide new strategies to enhance tumor clearance and resistance to disease.

Figure 1

Higher dose and longer duration of α-PD-L1 mAb treatment does not improve survival, suggesting resistance to α-PD-L1 mAb treatment. Mice were injected with K7M2 cells, and treated for either 15 days (n = 10) or 30 days (n = 10) with α-PD-L1 mAb. Survival was not significantly different between the 15 day and 30 day treatment, p = 0.2942, and all mice succumbed to disease (A). At time of necropsy, PD-L1, CD80, and CD86 expression was evaluated using Flow Jo. PD-L1 expression was significantly decreased in treated versus untreated mice, p = 0.0026. CD80 expression was significantly increased in treated versus untreated mice, p < 0.0001. CD86 expression was significantly increased in treated versus untreated mice, p < 0.0001 (B). At time of necropsy, PD-1+, CTLA-4+, and LAG3+ CD8+ expression was evaluated using Flow Jo. PD-1 + CD8+ expression was significantly different in treated versus untreated mice, p = 0.0005 (C). CTLA-4 + CD8+ expression was significantly different in treated versus untreated mice, p = 0.043 (D). LAG3 + CD8+ expression was not significantly different between treated and untreated mice (E).

Figure 2

Naïve mice injected with in vivo treated α-PD-L1 mAb K7M2 cells are non-responsive to α-PD-L1 treatment, suggesting resistance to α-PD-L1 treatment. Naïve Balb/cJ mice were injected with in vivo α-PD-L1 mAb treated metastatic osteosarcoma, with decreased PD-L1 expression, (B) black histogram is PD-L1 expression prior to reimplantation in comparison to mock treated mice in white, and treated with α-PD-L1 mAb or mock (A). No significant difference was seen between survival in treated (n = 10) and untreated (n = 10) mice injected with treated metastatic osteosarcoma (C). PD-L1 expression on metastatic osteosarcoma from mice injected with in vivo α-PD-L1 mAb treated metastatic osteosarcoma compared to control K7M2 injected mice was significantly decreased, p = 0.0026 (D). At time of necropsy, PD-1 + CTLA-4+, and LAG3+ CD8+ expression was evaluated using Flow Jo. PD-1 + CD8+ expression was significantly different in mice injected with in vivo α-PD-L1 mAb treated metastatic osteosarcoma compared to control K7M2 injected mice, p = 0.0017 (E). CTLA-4 + CD8+ expression was significantly different in in vivo α-PD-L1 mAb treated metastatic osteosarcoma injected mice compared to control, p = 0.0231 (F). LAG3 + CD8+ expression was not significantly different between these two groups (G).

Figure 3

Dual α-CTLA-4/α-PD-L1 mAb treatment can completely eradicate metastatic osteosarcoma. Survival of combinational treatment of implantable metastatic osteosarcoma with α-CTLA-4 and α-PD-L1 mAb was significantly higher than α-PD-L1 mAb treatment alone, p = 0.0177. Additionally, survival of combination treatment α-CTLA-4/α-PD-L1 mAb compared to α-CTLA-4 mAb treatment alone was significantly different with a p value of p < 0.0001. α-CTLA-4 mAb alone was not significantly different than mock treated mice. N = 10 for all treatment groups.

Figure 4

Dual α-CTLA-4/α-PD-L1 mAb treatment can elicit protective immunity towards metastatic osteosarcoma. To evaluate protective immune responses to tumor, cured α-CTLA-4/α-PD-L1 mAb mice (n = 12) were challenged with 10⁶ K7M2 cells at day 100. Cured α-CTLA-4/α-PD-L1 mAb treated mice cleared disease and significantly survived longer than age-matched control mice, p = 0.0049 (A). Lung tissue was evaluated for the presence of memory CD8 cells, and approximately 87% of the tumor-reactive memory T cells were CCR7 + CD62L+ (B). These CCR7 + CD62L + CD8+ cells were specific towards K7M2 cells used to generate the tumor, and able to produce IFNy and TNF in response to re-exposure, p=0.002 (C). To evaluate if protective immune responses to tumor were in fact due to memory CD8 T cells. Cured α-CTLA-4/α-PD-L1 mice (n = 6) were depleted of CD8 T cells at day 80, and challenged with 10⁶ K7M2 cells after depletion. Mice depleted of CD8 T cells prior to challenge all succumbed to metastatic osteosarcoma in the lung tissue, in comparison to 100% survival (n = 6) in cured immune competent mice challenged. A log-rank test gives a p = 0.0177 (D).

Figure 5

α-PD-L1 mAb treatment coupled with doxorubicin treatment does not enhance survival over α-PD-L1 mAb treatment alone. Survival of combinational treatment of metastatic osteosarcoma with doxorubicin and α-PD-L1 mAb was significantly higher than chemotherapy treated alone group, p = 0.0026, although no significant difference in survival was seen between combinational α-PD-L1 mAb and doxorubicin treatment and α-PD-L1 mAb alone. N = 10 for all treatment groups.