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D. Stroopinsky et al.
been disappointing in hematologic malignancies, including AML.26 It has been postulated that the lack of a significant intrinsic T-cell response in AML likely explained why there was an insufficient immune substrate for checkpoint inhibi- tion to be effective.
We have developed a personalized tumor vaccine in which patient-derived tumor cells are fused with autolo- gous DC so that a broad array of tumor antigens, including unique neo-antigens, are presented in the context of DC- mediated co-stimulation.4,5 In a phase II clinical trial, vacci- nation was associated with a dramatic and durable expan- sion of leukemia-specific T cells in the peripheral blood and marrow and was associated with more than 70% of patients remaining in remission with a mean follow-up of 5 years, despite the study patients having a mean age of 63 years.6 Of note, we demonstrated that leukemia-specific T cells were nearly absent in the peripheral blood and mar- row prior to vaccination. We postulated that DC/tumor fusions would create the necessary expansion of tumor-spe- cific T cells, which could then be further activated and expanded by the presence of checkpoint inhibition. We hypothesized that combined therapy with vaccination and checkpoint inhibition would be synergistic in providing effector cells that maintain a state of activation in the con- text of the immunosuppressive milieu of the tumor microenvironment.
In the present study, we established an immunocompe- tent murine AML model in which disease burden could be quantified and tracked by bioluminescence imaging. In this model of aggressive disease, we demonstrated that chal- lenge with syngeneic AML cells resulted in rapid engraft- ment and death of the host animals within 30 days. Consistent with prior clinical experience the introduction of checkpoint inhibition, via a mix of antibodies targeting PD- 1, TIM3, and RGMb, only modestly slowed disease pro- gression and did not significantly affect the survival of the mice. Of note, vaccination with syngeneic DC/AML cells 24 h after tumor challenge, at a state of minimal disease, markedly slowed disease progression and resulted in long- term survival in a significant subset of animals. As most AML patients with active disease have a deficient immune system, the ideal clinical setting for vaccination is in low tumor burden states, which are more favorable to allow priming of the immune response and induction of long- term memory. This has been demonstrated in subset analy- ses from several trials of cancer vaccines in which vaccine effectiveness was notably higher in patients with minimal or no residual disease.27
Remarkably, the combination of vaccine and checkpoint inhibition was uniquely capable of eradicating disease and producing long-term survival in all of the animals. In addi- tion, animals treated with the combination of vaccination and checkpoint inhibition were protected from leukemia engraftment after re-challenge with an otherwise lethal dose of leukemia cells, an effect consistent with generation of a memory response with long-term efficacy. The thera- peutic efficacy of vaccination and combined checkpoint blockade was largely reproduced by vaccination with PD-1 blockade alone, whereas vaccination with TIM3 blockade alone enhanced the initial response to the vaccine but resulted in only a subset of animals being resistant to re- challenge with the tumor. Interestingly, vaccination in com- bination with anti-RGMb did not affect the animals’ sur- vival in this model. This observation could in part be explained by the lack of PD-L2 expression by TIB-49 AML
tumor cells (data not shown), which would prevent signaling through that inhibitory pathway.
To further elucidate the immunological mechanism responsible for these observations, we studied the impact of therapy on leukemia-specific T-cell immunity in each of the differently treated cohorts of animals. Consistent with the underlying hypothesis, checkpoint inhibition did not increase the relative prevalence of leukemia-specific T cells above that present in untreated animals challenged with tumor cells. Of note, while vaccination with DC/AML fusions resulted in the expansion of leukemia-specific T cells in the peripheral blood and spleen, the levels of these cells were nearly doubled when animals were treated with the combination of vaccination and checkpoint inhibition. This demonstrated an important concept that while check- point blockade was not able to induce primary leukemia- specific immunity as a single modality, it was capable of further expanding vaccine-educated T cells. These findings were reproduced in an additional immunocompetent murine model of primary inducible IDH2 leukemia. Mice treated with a combination of a syngeneic fusion vaccine and checkpoint blockade demonstrated significantly decreased tumor burden as well as a 4-fold expansion of AML specific T cells.
Moreover, assessment of antigen-specific antitumor immunity revealed a significant increase in the frequency of survivin-specific T cells, as demonstrated by an increase in T cells positive for CD8 and survivin pentamers following vaccination and checkpoint inhibition compared to the numbers of these cells in controls. Interestingly, no change was observed in the frequency of cytomegalovirus-specific T cells, suggesting the tumor-specific nature of the T-cell activation and expansion.
The nature of the immune response following vaccina- tion and checkpoint blockade was further interrogated by single-cell RNA sequencing, which demonstrated compara- tively increased activation of signaling pathways regulating cell viability, proliferation and survival among effector and memory T cells, and downregulation of apoptosis-regulat- ing genes. Functional memory T cells have been shown to be a critical immune subset, with their presence being pre- dictive of more durable responses following immunothera- py.28 Furthermore, signaling pathway analysis within CD8 and CD4 memory T-cell compartments demonstrated sig- nificant upregulation of crucial pathways such as CD28 and TCR signaling, as well as mTOR signaling, known to be important in memory T-cell differentiation,29 following vac- cination as compared to the state in untreated controls. Interestingly, this effect was enhanced, with further upreg- ulation, following the addition of checkpoint blockade. Analysis of signaling in effector CD8/Nkg7 T cells showed significant upregulation of NF-κB, mTOR, CD28 and ICOS after vaccination, consistent with induction of an inflamma- tory phenotype.30-33 The combination of vaccination and checkpoint blockade further enhanced the activation of these signaling pathways.
In the context of enhanced expansion of tumor reactive lymphocytes and activation of TCR signaling pathways, we subsequently quantified the effect of vaccination in combination with checkpoint blockade on T-cell diversity repertoire. Vaccination with DC/AML fusions resulted in enhanced clonal diversity and the oligoclonal expansion of specific TCR sequences, consistent with vaccine-mediated targeting of tumor-associated epitopes. Importantly the addition of checkpoint inhibitors further enhanced this
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haematologica | 2021; 106(5)