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D. Stroopinsky et al.
AB
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. Naïve, 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.
Mice that had initially been challenged with tumor inoc- ulation and rendered disease-free by combined vaccine and checkpoint inhibitor therapy were subsequently re-chal- lenged via retro-orbital inoculation with a lethal dose (5×103) of lmTIB at day 90. Age-matched naïve C57BL/6J control mice were challenged with 5×103 lmTIB as controls. Mice were followed for survival and disease progression with bioluminescence imaging. In contrast to control ani- mals, mice that had been previously treated with the com- bination of vaccination and checkpoint inhibition were uni- formly protected from disease and showed no evidence of leukemic engraftment (Figure 4A and B).
We subsequently examined the relative contribution of each individual checkpoint inhibitor on vaccine efficacy. C57BL/6J mice were treated with syngeneic fusion vaccine in combination with anti-PD-1, anti-TIM3 or anti-RGMb monoclonal antibodies, as described above. At day 90 after tumor challenge, three out of five mice treated with the fusion vaccine alone were free of disease, replicating our previous findings. The addition of anti-RGMb did not increase the efficacy shown by that of the fusion vaccine alone, with two out of five animals surviving. However, vaccination in conjunction with PD-1 blockade or TIM3 blockade alone resulted in 100% of animals surviving with- out evidence of disease (Figure 5A and B).
Among the cohorts with complete response to treatment, i.e., those given vaccination and either anti-PD-1 (n=5) or anti-TIM-3 (n=5), mice were re-challenged with 50×103 lmTIB cells to evaluate long-term anti-leukemia immunity. Mice were followed for an additional 90 days, after which it was found that all five of the mice in the cohort given the vaccine plus atni-PD-1 remained disease-free after re-chal- lenge, whereas two of five mice in the TIM-3 cohort suc- cumbed to disease (Figure 5C).
Vaccination in conjunction with PD-1 blockade leads to an Increase in inflammatory signaling pathways
and blunts apoptosis of memory T cells
The nature of the immune response was further charac- terized by interrogation of the immune transcriptome fol- lowing vaccination in the context of PD-1 blockade as com- pared to checkpoint inhibition or vaccination alone.
Unsupervised analysis by single-cell RNA sequencing iden- tified comparable numbers of single-cell clusters in all three cohorts of mice (Figure 6A). Supervised analysis of CD4/IL7R and CD8/IL7R memory T cells demonstrated activation/upregulation in signaling pathways regulating T- cell viability, proliferation and survival after vaccination, which were further enhanced after treatment with both the fusion vaccine and checkpoint blockade. Consistent with these findings, there was significant downregulation in apoptosis-regulating genes (Figure 6B). A similar pattern was observed in CD8/Nkg7-expressing effector T cells with increases in viability, activation and proliferation and downregulation of apoptosis genes (Figure 6B).
Furthermore, signaling pathway analysis in the CD8/CDIL7R and CD4/CDIL7R memory compartment demonstrated significant effects on mTOR, CD28 and TCR signaling following vaccination compared to untreated con- trols. Interestingly, these signaling pathways were further upregulated following the addition of checkpoint blockade (Figure 6C and D). Analysis of signaling in effector CD8/Nkg7 cells showed significant upregulation of NFκB, mTOR, CD28 and ICOS signaling after vaccination, consis- tent with induction of an inflammatory phenotype. Combination treatment with the vaccine and anti-PD-1 fur- ther enhanced the activation of these signaling pathways (Figure 6E). CD8/Nkg7+ effector T cells also showed a sig- nificant impact on TCR signaling pathways (-log P value= 3.9); this effect was further enhanced (-log P value= 4.7) by addition of checkpoint blockade (Figure 6E).
Vaccination in combination with checkpoint blockade results in greater clonal T-cell diversity
The effect of vaccination alone or in conjunction with checkpoint blockade on the T-cell repertoire was interrogat- ed by clonotypic analysis using targeted TCR profiling stud- ies. The diversity index and rarefaction analysis demon- strated that vaccination resulted in the selective expansion of clonal populations and enhanced diversity of the T-cell repertoire compared to peripheral blood samples derived from untreated control mice (Figure 7A and B). Remarkably, diversity was significantly further expanded by sequential therapy with DC/AML fusions and combined
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