A Novel Monoclonal Antibody Combination Plus DC/AML Fusion Vaccine Eradicates AML in an Immunocompetent Murine Model
Authors: Stroopinsky Dina , Liegel Jessica , Mahoney Kathleen M. , Rosenblatt Jacalyn , Ebert Benjamin L. , Stone Richard M. , Rahimian Maryam , Nahas Myrna , Orr Shira , Capelletti Marzia , Weinstock Matthew , Jain Salvia , Bisharat Lina , Ghiasuddin Haider , Morin Adam , Kufe Donald , Freeman Gordon J , Avigan David E. 
Published: October 2019

Abstract
Introduction: Acute myeloid leukemia (AML) is a lethal hematologic malignancy in which standard therapy is often inadequate in sustaining durable remissions. In part due to an immunosuppressive milieu characterized by the upregulation of checkpoint inhibitory pathways leading to T cell anergy, ineffective antigen (Ag) presentation, and increased numbers of myeloid derived suppressor cells (MDSCs). We have pioneered a novel vaccine that targets an array of leukemic antigens by fusing whole patient-derived AML cells to autologous dendritic cells (DCs) in the presence of polyethylene glycol (PEG). In a phase I/II clinical trial in AML, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Checkpoint blockade is being investigated in combination with cancer vaccines to overcome the suppressive effects of the tumor microenvironment on cytotoxic tumor-specific T cells. Here we describe the addition of a novel combination of checkpoint inhibitors with DC/AML fusion vaccine in an immunocompetent murine model.
Methods/Results: C57BL/6J mice underwent retro-orbital inoculation with 50,000 syngeneic luciferase and mCherry labeled TIB-49 AML cells. 24 hours later a cohort of mice was treated with 100,000 DC/AML irradiated fusion cells. An additional cohort was treated with 6 doses of a combination of anti-PD1/anti-TIM3/anti-RGMb mAbs administered IP every 3 days with and without vaccine. Control mice were treated with appropriate isotype control. AML burden was evaluated in peripheral blood (PB) 14 days post inoculation using flow cytometric analysis for mCherry expression. AML cells were present in PB of control mice and mice treated with check point inhibitors alone (mean 1.2% and 1.5% respectively). Lower disease burden was detected in mice treated with the DC/AML fusion vaccine (0.5%) or combination of fusion vaccine with check point inhibitors (0.5%).
Furthermore, to measure tumor-specific T cell response, PB cells were cultured in the presence of autologous TIB-49 tumor lysate for 3 days and analyzed for intracellular IFN-γ expression using multicolor flow cytometric analysis. Mice treated with combination of DC/AML fusion vaccine and checkpoint inhibition showed a significant increase in IFN-γ expression by CD8+ T cells with mean values of 6.4%. Mice treated with vaccine, checkpoint inhibitors alone or IgG control demonstrated mean levels of 3.4%, 1.9% and 1.9% respectively (n=5; p=0.01).
Additional immunologic assessment was performed from PB of surviving animals 37 days after tumor challenge. Combination treatment with DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs led to a statistically significant increase in CD4/CD44+/CD62L- memory T cells with concurrent decrease in naïve CD8/ CD44-/CD62L+ T cells as compared to single agent treatments. Furthermore, a statistically significant decrease in CD4+CD25+FOXP3+ Tregs was detected after combination treatment compared to single treatment groups.
The mice were followed for survival and disease progression using BLI imaging. All control mice developed visible AML by luminescence following luciferin injection as well as symptomatic disease requiring euthanasia by day 31 after initial challenge with tumor cells (Figure 1). Mice treated with the anti-PD1/anti-TIM3/anti-RGMb mAbs alone demonstrated a survival benefit but all required euthanasia by day 44. 3 of 5 mice treated with vaccine alone remain disease free at more than 65 days. And strikingly, the entire cohort of mice treated with the combination of DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs remain alive and disease free in this aggressive AML model.
Conclusion: In the current study we have demonstrated the capacity of a combination of PD-1, TIM-3 and RGMb checkpoint inhibition to create an enhanced environment for immune response to the DC/AML fusion vaccine in an immunocompetent murine AML model. Treatment with this combination led to an increase in tumor specific T cell immunity, decrease in circulating Tregs and shift toward a memory phenotype. Most significantly, mice who received the combination treatment remain disease-free several months post inoculation. This synergistic approach has promising translational potential and a phase 1 clinical trial is planned.
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Disclosures
Rosenblatt: Bristol-Myers Squibb: Research Funding; Merck: 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; Celgene: Research Funding. Stone:Astellas: Consultancy; Otsuka: Consultancy; Arog: Consultancy, Research Funding; Argenx: Other: Data and Safety Monitoring Board; Pfizer: Consultancy; Fujifilm: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Orsenix: Consultancy; Amgen: Consultancy; Merck: Consultancy; Agios: Consultancy, Research Funding; AbbVie: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Cornerstone: Consultancy; Ono: Consultancy; Sumitomo: Consultancy. Freeman:Roche: Patents & Royalties; Merck: Patents & Royalties; Origimed: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Patents & Royalties; Xios: 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; Novartis: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Roche: Membership on an entity's Board of Directors or advisory committees; EMD-Serono: Patents & Royalties; AstraZeneca: Patents & Royalties.