Ex-Vivo Stimulation with DC/AML Fusion Vaccine in the Presence of Cytokines Leads to an Activated T Cell Memory Phenotype and Enhanced Cytotoxicity with Potential for Use As an Adoptive Cellular Therapy
Authors: Liegel Jessica , Stroopinsky Dina , Mendez Lourdes M. , McMasters Malgorzata , Ghiasuddin Haider , Rahimian Maryam , Nahas Myrna , Weinstock Matthew , Jain Salvia , Capelletti Marzia , Bisharat Lina , Morin Adam , Orr Shira , Rosenblatt Jacalyn , Kufe Donald , Avigan David E. 
Published: October 2019

Abstract
Introduction: Adoptive cellular transfer using engineered cells in hematologic malignancies often rely on single antigens as targets but antigen downregulation has been described as a mechanism of tumor resistance. Furthermore, identification of optimal tumor-specific antigens that are immunogenic and have tolerable off-target effects remain an area of active investigation. Our group has developed a novel vaccine using whole patient-derived AML cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. Here we describe the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo and provide a framework for therapeutic exploration of vaccine stimulated T cells as a cellular therapy.
Methods/Results:
Vaccine was generated with C57BL/6J bone marrow matured DCs and syngeneic TIB-49 AML cells as previously described. T cells were obtained from C57BL/6J splenocytes after magnetic bead isolation and cultured with vaccine plus IL-15 and IL-7. Vaccine stimulated T cells showed increased immune activation as measured by multicolor flow cytometric analysis. Compared to unstimulated T cells, there was 8-fold increase in CD3+CD69+ expression, which is detected upon TCR ligation. Likewise, there was a 12-fold and 3-fold increase in CD137 expression on CD4+ and CD8+ cells, which is up-regulated upon antigen recognition.
In addition, a shift from naïve to memory T cell phenotype occurred, which has important implications for response to adoptive cell transfer. Amongst CD4+ cells, the CD44+CD62L- subset comprised 71% after vaccine stimulation. Amongst CD8+ cells, there was increase in both effector and memory subsets. Furthermore, there was enhanced Th1 polarization in ex-vivo culture with vaccine, as IFN-γ expression was increased 2-fold and 5-fold in CD4+ and CD8+ subsets respectively. A corresponding 1.5-fold increase in cytotoxicity was detected using a standard granzyme B CTL assay.
The efficacy of adoptive therapy with vaccine stimulated T cells was demonstrated in a xenograft model in which NSG mice were engrafted with patient derived AML cells following sublethal total body irradiation. T cells autologous to the primary tumor were obtained at the time of disease remission and DC/AML fusions were generated. T cells were cultured with or without fusion vaccine at 1:10 ratio. Intracellular IFN-γ expression increased 9-fold and 11-fold on CD8+ and CD4+ cells respectively with ex-vivo vaccine stimulation compared to unstimulated T cells. On day 14 after tumor inoculation, mice were injected with 1.5 x 106 T cells that had been cultured with or without fusion vaccine. Untreated mice were used as a control. The mice were sacrificed two weeks later at which time bone marrow and spleen cells were harvested.
AML engraftment in bone marrow was significantly reduced in mice treated with ex -vivo vaccine stimulated T cells compared to control as measured by presence of hCD45+hCD33+ leukemia cells with mean levels of 18% and 38% respectively (n=4). Increased marrow infiltration of human CD8+ T cells was detected in animals treated with ex vivo stimulated T cells and corresponded with lower AML burden. In contrast, both control mice and those given unstimulated T cells demonstrated low hCD8+ marrow T cell infiltration and higher disease burden. We propose this was due to tumor-specific T cell activation of vaccine educated T cells as evidenced by their increased levels of hCD8+IFN-γ expression following exposure to autologous tumor lysate at the time of splenocyte harvesting.
Conclusion: In the current study we have shown the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo, as demonstrated by increased CD69, CD137 and IFN-γ expression, and enhance memory and effector subsets when co-cultured in the presence of cytokines. Results of treatment with vaccine stimulated T cells to date have shown reduced engraftment of primary AML in NSG mice. This provides a framework to evaluate the therapeutic use of adoptive transfer of ex-vivo fusion vaccine stimulated T cells in AML. Further in vitro work incorporates marrow-infiltrating T cells and circulating tumor-specific T cells to assess efficacy in active disease. In vivo models are also ongoing including both xenograft and an immunocompetent murine model and subsequent survival analyses will be reported.

Disclosures
Rosenblatt: Celgene: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding.