Leukemia vaccine overcomes limitations of checkpoint blockade by evoking clonal T cell responses in a murine acute myeloid leukemia model

Abstract

We have developed a personalized vaccine whereby patient derived leukemia cells are fused to autologous dendritic cells, evoking a polyclonal T cell response against shared and neo-antigens. We postulated that the dendritic cell (DC)/AML fusion vaccine would demonstrate synergy with checkpoint blockade by expanding tumor antigen specific lymphocytes that would provide a critical substrate for checkpoint blockade mediated activation. Using an immunocompetent murine leukemia model, we examined the immunologic response and therapeutic efficacy of vaccination in conjunction with checkpoint blockade with respect to leukemia engraftment, disease burden, survival and the induction of tumor specific immunity. Mice treated with checkpoint blockade alone had rapid leukemia progression and demonstrated only a modest extension of survival. Vaccination with DC/AML fusions resulted in the expansion of tumor specific lymphocytes and disease eradication in a subset of animals, while the combination of vaccination and checkpoint blockade induced a fully protective tumor specific immune response in all treated animals. Vaccination followed by checkpoint blockade resulted in upregulation of genes regulating activation and proliferation in memory and effector T cells. Long term survivors exhibited increased T cell clonal diversity and were resistant to subsequent tumor challenge. The combined DC/AML fusion vaccine and checkpoint blockade treatment offers unique synergy inducing the durable activation of leukemia specific immunity, protection from lethal tumor challenge and the selective expansion of tumor reactive clones.

Figure 1.

Checkpoint blockade does not significantly affect acute myeloid leukemia engraftment in vivo. C57BL/6J mice were retroorbitally inoculated with 50x10³ syngeneic TIB-49 acute myeloid leukemia cells that were stably transduced with luciferase/mCherry. The mice were then treated with six doses of 200 μg anti-PD1, anti-TIM3, or anti-RGMb or a combination (combo) of the three monoclonal antibodies using intraperitoneal injections every 3 days. Some mice were treated with appropriate isotype control as a negative control. To determine the progression of the leukemia, (A) bioluminescence imaging of each group of mice (n=5) was performed on days 17 and 21 after inoculation with the tumor and (B) the mice were followed for survival for 90 days. The results are shown in a Kaplan-Meier curve.

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 treated 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.

Figure 3.

Increase in tumor-specific T cells following combination treatment with the fusion vaccine and PD1/TIM3/RGMb blockade. C57BL/6J mice were treated as described in Figure 2. (A, B) On day 14 peripheral blood was collected and CD8⁺ T cells were assessed for intracellular interferon-gamma (IFNγ) expression using multichannel flow cytometry following exposure to autologous tumor lysate for 3 days. Results are presented as a summary of data from the five mice in each group (**P<0.05) (A) and representative dot plots (B). (C-E) In a similar independent experiment on day 17 after tumor challenge, the mice underwent bioluminescence imaging analysis (C). The mice were then euthanized; splenocytes were harvested and assessed for IFNγ expression using multichannel flow cytometry following exposure to autologous tumor lysate. Results are presented as a summary of data from the five mice in each group (***P<0.05) (D) and as representative dot plots (E). (F) Spleen-derived CD8⁺ T cells were also assessed for frequency of tumor antigen-specific T cells with multichannel flow cytometry using pentamer analysis. TIB-49 acute myeloid leukemia (AML) cells were confirmed to express survivin using intracellular flow cytometric analysis; unstained cells and cells incubated with an appropriate isotype control were used as negative controls. Binding of APC-labeled multimeric MHC/survivin peptide complexes to T-cell receptors was examined to determine the frequency of the survivin-specific T cells. (G, H) The frequency of survivin-specific T cells is presented in gated CD8⁺ T cells as a summary of data from the five mice in each group (***P<0.05) (G) and as representative dot plots (H). The frequency of cytomegalovirus-specific T cells was analyzed as a control. C57BL/6J mice were inoculated by tail vein injection with 200x10³ primary syngeneic mutant IDH2 AML cells that were stably transduced with green fluorescence protein (GFP). Syngeneic DC/mIDH2 AML fusion cells were generated. The mice were then treated with either vaccine alone, anti-PD1/TIM3/RGMb or a combination of anti- PD1/TIM3/RGMb and the fusion vaccine. Some mice were treated with an appropriate isotype control as a negative control. (I, J) Disease burden was assessed in spleens of animals at day 36 after tumor challenge by detection of the GFP signal via flow cytometric analysis. Results are presented as a summary of data from the five mice in each group (***P<0.05) (I) and as representative histograms (J). On day 14 peripheral blood was collected and CD8⁺ T cells were assessed for intracellular IFNγ expression using multichannel flow cytometry following exposure to autologous tumor lysate for three days. Results are presented as (K) a summary of 5 analyzed mice (n=5; P<0.05) and (L) as representative dot plots.

Figure 4.

Treatment with the fusion vaccine in combination with PD1/TIM3/RGMb blockade prevents establishment of acute myeloid leukemia upon re-challenge with tumor cells. Ninety days after the initial tumor challenge and treatment with six doses of anti-PD1/TIM3/RGMb (mAbs), C57BL/6J mice (n=5) were challenged with an additional dose of 50,000 luciferase/mCherry-transduced syngeneic TIB-49 cells. Naive, age-matched mice (n=5) were inoculated with the same dose as controls. To assess AML burden, (A) bioluminescence imaging was performed after re-challenge and (B) the mice were followed for survival for 90 days, as illustrated in a Kaplan-Meier curve.

Figure 5.

Combination treatment with the fusion vaccine + anti-PD1 or fusion vaccine + anti-TIM3 prevents the 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. Syngeneic dendritic cell (DC)/AML fusion cells were generated. The mice were then treated with either vaccine alone, or with fusion vaccine in combination with anti-PD1; anti-TIM3 or anti-RGMb. Control mice were treated with the appropriate isotype control. To assess the progression of AML, (A) bioluminescence imaging was performed starting on day 30 after inoculation and (B) the mice were followed for survival as illustrated in the Kaplan Meier curve (n=5 in each group). At 90 days after the initial tumor challenge, mice treated with vaccine + anti-TIM3; and vaccine + anti-PD-1 were re-challenged with an additional dose of 50x10³ syngeneic TIB-49 AML cells. (C) The mice were followed for survival for another 90 days. The results are shown in a Kaplan Meier curve.

Figure 6.

Single-cell RNA sequencing analysis demonstrates that vaccination alone and in combination with checkpoint inhibition affects the T-cell landscape. (A) Singlecell RNA-sequencing analysis of peripheral blood mononuclear cells isolated from control mice, mice treated with the dendritic cell (DC)/acute myeloid leukemia (AML) fusion vaccine, and mice treated with the vaccine + checkpoint point inhibitors. (n=3 mice/group). Visualization of single-cell clusters was achieved using the UMAP approach from normalized data of 710 control, 884 vaccine-treated and 1,489 combination-treated peripheral blood mononuclear cells. Cell clusters were annotated based on expression of established immune-cell markers (e.g., T cells [CD3+], B cells [CD19⁺], memory T cell [Il7r⁺], and effector cells [Sell⁺, CD62L^–] (left panel). Relative proportions of cells in the clusters from each cohort are depicted with different colors (right panel). (B) Functional enrichment heatmap depicting increased (red) or decreased (green) functional categories in the samples from animals treated with vaccine alone or the combination (combo). The heatmap was prepared based on zscores calculated using ingenuity pathways analysis systems. (C-E) Pathways that are significantly affected in various subsets of memory T cells: CD8a and Il7r⁺ T cells (C), CD4 and Il7r⁺ T cells (D), and effector T cells (E). Black and gray bars represent the significance of the impact of vaccine alone and in combination with checkpoint inhibition, respectively, on selected signaling pathways. The extent of activation/increase of various significantly affected pathways is shown using overlapping orange bars.

Figure 7.

Vaccination with the fusion vaccine leads to greater clonal T-cell diversity, which is further enhanced following checkpoint blockade. C57BL/6J mice were treated as described in Figure 6. Peripheral blood (PB) was then collected and assessed for T-cell diversity using targeted T-cell receptor (TCR) profiling. (A) Inverse Simpson diversity index indicating that vaccine alone and in combination with checkpoint inhibitors enhance T-cell diversity (B) Refraction diversity analysis. (C) Bubble plot of the expression of the top TCR clones after vaccination alone or in combination with checkpoint inhibitors. Columns represent samples and rows represent amino acid sequences of different TCR clones. The TCR clones with significant increases or decreases are represented by red and green, respectively. The fold-change of the top ten TCR clones for each sample is calculated compared to the untreated control samples.