Page 113 - 2021_05-Haematologica-web

Vaccine overcomes checkpoint inhibitor limitation
strated a modest improvement in survival compared to control animals but all required euthanasia by day 44. The vaccine alone prevented leukemic engraftment in the majority of animals, with two of five mice remaining free of disease at 90 days. Remarkably, the entire cohort of mice treated with vaccination and checkpoint blockade remained alive and disease-free in this model of aggressive AML 90 days after inoculation (Figure 2C).
Checkpoint inhibition in conjunction with a dendritic cell/acute myeloid leukemia fusion vaccine leads
to an increase in tumor-specific immunity
We subsequently examined whether the combination of vaccination with checkpoint inhibition would result in enhanced AML-specific immunity in vivo. To measure tumor-specific T-cell responses, peripheral blood cells were collected from mice 14 days after inoculation and tumor recognition was assessed in a modified enzyme-linked immunospot assay in which the percentage of CD8 T cells exhibiting intracellular IFN-γ expression after 3 days of stimulation with autologous TIB-49 tumor lysate was quantified. Mice treated with checkpoint inhibitors alone showed no expansion of T cells expressing IFN-γ following exposure to autologous tumor lysate as compared to con- trol animals which showed mean percentages of 1.9% for both groups (n=5). Mice treated with the fusion vaccine
A
showed variable expansion of tumor reactive T cells with a mean level of IFN-γ expression of 3.4% (n=5; P=ns). This mean expansion did not meet statistical significance because of the variability in tumor-specific T cells in responding and non-responding animals. However, mice treated with both the fusion vaccine and checkpoint inhibi- tion showed a significant expansion of circulating tumor- specific CD8+ T cells with a mean value of IFN-γ-expressing cells of 6.4% (n=5; P=0.01) (Figure 3A and B).
In a subsequent experiment, the enhanced expansion of tumor-specific T cells following vaccination and checkpoint inhibition was confirmed in splenocyte populations in ani- mals euthanized 17 days after tumor challenge. Consistent with our prior study, combination therapy resulted in the most pronounced induction of tumor-specific immunity associated with the prevention of leukemia engraftment in all of the treated animals (Figure 3C-E).
To further elucidate the capacity of fusion cell vaccination and checkpoint blockade to elicit tumor-specific immunity in vivo, we quantified antigen-specific T cells targeting sur- vivin following treatment with a checkpoint inhibitor, vac- cination or combination therapy. Survivin is a member of the Inhibitor of apoptosis family, known to be overex- pressed in AML. Using intracellular flow cytometric analy- sis, we demonstrated high levels of survivin expression by TIB-49 cells (Figure 3F). Expansion of survivin-specific T
Figure 2. Combination treatment with the fusion vaccine and PD1/TIM3/RGMb blockade prevents establishment of acute myeloid leukemia in vivo. C57BL/6J mice were retro-orbitally inoculated with 50x103 syngeneic TIB-49 acute myeloid leukemia (AML) cells that were stably transduced with luciferase/mCherry. (A) Syngeneic dendritic cell (DC)/AML fusion cells were generated as described and evaluated for co-expression of tumor (mCherry) and DC (CD86) markers using flow cytometry. The mice (n=5 in each group) were then treated with either vaccine alone, anti-PD1/TIM3/RGMb (mAbs) or a combination of anti- PD1/TIM3/RGMb and the fusion vaccine. One group of mice was treat- ed with appropriate isotype control as a negative control. (B) Bioluminescence imaging was performed serially starting on day 29 after inoculation (3 representative mice are shown). (C) The mice were followed for survival for 90 days. The results are shown in a Kaplan Meier curve.
C
B
haematologica | 2021; 106(5)
1333