Introduction: Immunotherapy for AML holds promise in overcoming chemotherapy resistance and in preserving immunologic memory necessary for durable remissions.A bispecific T-cell engaging antibody targeting CD3 and CD123 (CD123-CODV-TCE) has been shown to stimulate T cells to target CD123-expressing leukemic cells in vitroand in mouse models 1. While the short-term immune stimulation mediated by the CD123TCE has the potential to result in clinical response, long-term disease control will require the development of immune memory. We have developed a personalized cancer vaccine in which patient's dendritic cells are fused with autologous leukemia cells resulting in presentation of a wide range of antigens to the immune system. Here, we describe a novel combination of CD123TCE with a DC/AML fusion vaccine ex vivo and in a xenograft murine model. We hypothesized that the CD123TCE will direct the vaccine-educated T cells to not only more effectively eradicate target leukemia cells but also evoke a repertoire of memory T cells and long-term response.
Methods/Results: AML cells expressing CD123 were isolated from bone marrow mononuclear cells (BMMCs) from AML patients (n=3). DCs were generated from autologous adherent peripheral blood mononuclear cells (PBMCs) obtained at the time of disease remission as previously described 2,3. Concurrently, CD3+ T cells were isolated from the non-adherent fraction of PBMCs using magnetic bead separation. Fusion cells were generated by co-culturing the DC and tumor cells at a ratio of 3:1 in the presence of polyethylene glycol (PEG). Vaccine educated T cells were then generated by co-culture of T cells with the autologous fusion cells at a ratio of 10:1 for 5-7 days followed by T cell expansion via CD3/CD28 ligation.
The capacity of the vaccine-educated T cells to target autologous leukemia cells with the addition of CD123TCE was assessed. The results demonstrated a statistically significant increase in Granzyme B activity in the target AML cells following co-culture with vaccine-educated T cells and the addition of the CD123TCE, compared to T cells + isotype control (n=3). Furthermore, vaccine stimulation in combination with CD123TCE led to a robust increase in induction of tumor specific activated T cells as detected by CD137 expression and intracellular IFN-γ production after co-culture of vaccine-educated T cells with autologous tumor cells in the presence of CD123TCE. The addition of CD123TCE to vaccine-educated T cells resulted in mean 25.4% and 9.6% intracellular IFN-γ expression for CD8 and CD4 T cells, respectively, compared to 8.5 and 3.1% IFN-γ expression following the addition of isotype control (n=3).
Next, we examined the efficacy of the combined treatment with vaccine-educated T cells and CD123TCE in-vivo, in two independent xenograft experiments. NSG mice were irradiated with 300rads and challenged with 1x10 6 patient-derived CD123+ tumor cells via retro-orbital injections. After detection of human AML engraftment in the PB on day 76, the mice were inoculated IV with 1X10 6 resting, or ex vivo fusion vaccine educated autologous T cells IV. Subsequently, cohorts of mice were treated with CD123TCE or an appropriate isotype control every 3 days IP. A significant decrease in human leukemia burden was detected in the peripheral blood, spleen and bone marrows of analyzed animals after treatment with vaccine educated T cells and isotype control, or resting T cells and CD123TCE, compared to untreated mice (n=5). Strikingly, no detectable AML was found in peripheral blood, spleens and bone marrows of mice treated with vaccine educated T cells in combination with TCE (n=5). Of note, treatment with vaccine educated T cells led to an expansion of human CD3+ T cells in tissues obtained from the analyzed animals. These human T cells persisted in mice treated with the CD123TCE with a two-fold increase in tumor-specific CD8+ T cells, as assessed by intracellular IFN-γ secretion following ex vivo stimulation with autologous tumor lysate.
Conclusions: We demonstrated that the combination of DC/AML fusion vaccine and CD123TCE led to increase in tumor specific T cell immunity, both ex-vivo and in a xenograft murine model when compared to uneducated T cells with CD123TCE or educated T cells with isotype control molecule. Most significantly, the combination treatment was shown to eradicate AML in this model with all animals remaining disease-free several months post inoculation.
Stroopinsky: The Blackstone Group: Consultancy. Nahas: Kite Pharma: Current Employment. Fraenkel: Sanofi: Current Employment. Yildirim: Sanofi: Current Employment. Bonnevaux: Sanofi: Current Employment. Guerif: Sanofi: Current Employment. Kufe: Genus Oncology: Current equity holder in publicly-traded company; Canbas: Consultancy; REATA: Consultancy, Current equity holder in publicly-traded company; Hillstream BioPharma: Current equity holder in publicly-traded company. Rosenblatt: Parexel: Consultancy; Wolters Kluwer Health: Consultancy, Patents & Royalties; Bristol-Myers Squibb: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imaging Endpoints: Consultancy; Attivare Therapeutics: Consultancy. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.