A Blood Dendritic Cell Vaccine for Acute Myeloid Leukemia Expands Anti-Tumor T Cell Responses at Remission

ONCOIMMUNOLOGY 2018, VOL. 7, NO. 4, e1419114 (11 pages) https://doi.org/10.1080/2162402X.2017.1419114 ORIGINAL RESEARCH A blood dendritic cell vaccine for acute myeloid leukemia expands anti-tumor T cell responses at remission Jennifer L. Hsua, Christian E. Bryanta,b, Michael S. Papadimitriousa,c, Benjamin Konga,c, Robin E. Gasiorowskia, Daniel Orellanab, Helen M. McGuire d,e, Barbara Fazekas de St Groth d,f, Douglas E. Joshuab,c, P. Joy Hob,c, Stephen Larsenb, Harry J. Ilandb, John Gibsonb,c, Georgina J. Clarka,c, Phillip D. Fromm a,c, and Derek NJ Harta,c aDendritic Cell Research Group, ANZAC Research Institute, Sydney, NSW, Australia; bInstitute of Haematology, Royal Prince Alfred Hospital, Sydney, NSW, Australia; cDiscipline of Internal Medicine, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia; dRamaciotti Facility for Human Systems Biology, Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia; eMelanoma Immunology and Oncology Unit, Centenary Institute, The University of Sydney, Sydney, Australia; fDiscipline of Pathology, Sydney Medical School, The University of Sydney, Sydney NSW, Australia ABSTRACT ARTICLE HISTORY Only modest advances in AML therapy have occurred in the past decade and relapse due to residual Received 1 September 2017 disease remains the major challenge. The potential of the immune system to address this is evident in the Revised 11 December 2017 success of allogeneic transplantation, however this leads to considerable morbidity. Dendritic cell (DC) Accepted 12 December 2017 vaccination can generate leukemia-specific autologous immunity with little toxicity. Promising results KEYWORDS have been achieved with vaccines developed in vitro from purified monocytes (Mo-DC). We now Acute Myeloid Leukemia; demonstrate that blood DC (BDC) have superior function to Mo-DC. Whilst BDC are reduced at diagnosis Blood Dendritic Cell; in AML, they recover following chemotherapy and allogeneic transplantation, can be purified using CMRF- Immunotherapy; Allogeneic 56 antibody technology, and can stimulate functional T cell responses. While most AML patients in Haematopoietic Stem Cell remission had a relatively normal T cell landscape, those who had received fludarabine as salvage therapy Transplantation; have persistent T cell abnormalities including reduced number, altered subset distribution, failure to Chemotherapy; T Cell expand, and increased activation-induced cell death. Furthermore, PD-1 and TIM-3 are increased on CD4T Landscape; Checkpoint Inhibitor cells in AML patients in remission and their blockade enhances the expansion of leukemia-specific T cells. This confirms the feasibility of a BDC vaccine to consolidate remission in AML and suggests it should be tested in conjunction with checkpoint blockade. Introduction DC.9-11 BDC present RNA encoded antigen more effectively Acute myeloid leukemia (AML) is the most common adult than Mo-DC,9 stimulate better allogeneic T cell IFN-g acute leukemia. Intensive chemotherapy can induce complete responses,11 induce stronger CD4T cell proliferation11 and have remission (CR) but the majority of patients relapse. Removing superior migratory capacity.9,12 However, the rarity of BDC residual disease after conventional chemotherapy is a major (<1% of leukocytes) has limited their use in clinical vaccines. C C challenge. The potential of immune therapy to address this is A mixture of BDC including CD1c and CD141 myeloid demonstrated by the clinical efficacy of allogeneic haemato- DC (mDC), the most potent cross-presenting DC,13 can be puri- poietic cell transplant (ATx), which provides a graft-versus- fied in a single-step immune selection using a chimeric monoclo- leukemia (GVL) response through donor T cells. Natural nal antibody (mAb), hCMRF-56 mAb,9,14 andloadedwith C anti-AML immunity is demonstrated by the success with antigen to expand functional antigen-specificCD8 Tcells.9,12,14 checkpoint inhibitor therapy in elderly AML patients.1 Enhanc- We sought to determine if this approach were applicable in ing this T cell immunity is the goal of dendritic cell (DC) AML patients in CR after chemotherapy or ATx, when disease vaccination. burden is low and there is an opportunity to target residual dis- DC trials performed to date for AML have used “first genera- ease after haematological recovery. We determined that BDC are tion” vaccines, predominantly composed of manufactured deficientatdiagnosisbutreturnatCR,canbepurified using monocyte-derived DC (Mo-DC) resulting in detectable expan- hCMRF-56 mAb and drive the in vitro expansion of autologous sions of tumor antigen-specific T cell responses and some clini- viral and Wilms tumor 1(WT1) specific T cell responses. We cal efficacy.2-5 The use of pre-formed blood DC (BDC) as a tool add that whilst the T cell landscape is not altered in AML for DC vaccination has begun to garner interest,6 with develop- patients after standard chemotherapy, patients who have received ment of new methodologies for their isolation and reporting of fludarabine show persistent abnormalities associated with an landmark Phase I clinical trials.7,8 There are significant tran- inability to respond to vaccination. Finally, we describe the scriptional and functional differences between BDC and Mo- expression of immune checkpoint molecules by T cells from CONTACT Professor, Derek NJ Hart [email protected] ANZAC Research Institute, Gate 3 Hospital Road, Concord NSW 2139 Australia. Supplemental data for this article can be accessed on the publisher’s website. © 2018 Taylor & Francis Group, LLC e1419114-2 J. L. HSU ET AL. AML patients in CR, providing a rationale to combine BDC vac- Next we validated our gating strategy using ViSNE,22 which cination with checkpoint blockade, and demonstrate that this allowed us to directly compare healthy and patient BDC popula- can enhance BDC-induced tumor antigen-specificresponses. tions by visualising high dimensional data structures in two dimen- sions (Fig. 2B). All orthodox BDC subsets were identified in HD and AML CR patients but were reduced in active AML (Fig. 2B). Results We then performed a Trucount analysis23 assessing AML BDC are superior to Mo-DC at processing and cross- patients (patient characteristics Table S1) at new diagnosis (ND, presenting long peptide antigen n D 8), relapse (RR, n D 6), in CR post-chemotherapy (CT, n D 18) or >12 months post-ATx (ATx, n D 7) along with age- Cross-presentation of antigen is a fundamental function of DC as matched healthy controls (HD, n D 15, Fig 2C). This revealed C C professional antigen presenting cells, presenting exogenous pro- reduced numbers of CD1c mDC, CD141 mDC and plasmacy- tein as peptide antigen in the context of HLA class I. We assessed D < D C toid (pDC) at diagnosis (p 0.0001, p 0.0001, p 0.0013) and the capacity of Mo-DC and CD1c mDC (as they are the most at relapse (p < 0.0001, p < 0.0001, p < 0.0001). Within 12 weeks numerous DC in the CMRF-56 vaccine) to cross-present antigen. C C C following chemotherapy, CD1c mDC recovered and CD141 Both Mo-DC and CD1c mDC showed sustained presentation of mDCandpDCshowedpartialrecovery(pD 0.02 and p D 0.009 the short, surface loaded FMP58–66 peptide, in the context of HLA C respectively) when compared to HD. In four patients with AML- class I after 16 hours of culture (Fig. 1A, B). CD1c mDC were specific molecular abnormalities (NPM1 and FLT3-ITD), there fi able to present the peptide at signi cantly higher density than was no detectable molecular disease in the peripheral blood when < C Mo-DC (p 0.0001). Most notably, CD1c mDC had a strik- BDC recovered at CR, confirming that BDC do not contain the ingly increased capacity to cross-present a long FMP54–74 peptide molecular marker and are independent of the leukaemic clone. into short FMP58–66 HLA-A2 complexes at 16 hours compared with Mo-DC (Fig. 1A, B;pD 0.029). CMRF-56C BDC can be purified from AML patients in CR BDC are depleted at diagnosis but return in CR We confirmed that CMRF-56 antigen is not expressed by AML Markers used to identify BDC including HLA-DR, CD11c, CD123 blasts and does not up-regulate following culture, in contrast to and ILT3, are often present on leukemic blasts, and standard Line- its up-regulation by BDC from HD or AML patients in CR ¡ C C age HLA-DR gating may be insufficient to identify BDC in (n D 4 Fig. 3A). Up-regulation of CMRF-56 antigen by CD1c patients with circulating blasts, resulting in over-estimation of mDC from AML CR patients (n D 18) was similar to age- BDC levels in AML patients at diagnosis.15-21 To investigate this, matched HD (n D 14, p D 0.82, Fig. 3B). CD1c and CD141 ¡ C we sorted Lineage HLA-DR cells from three patients with circu- BDC upregulated CMRF-56 similarly (Figure S2). This was lating blasts and analysed their morphology. The great majority of accompanied by CD83 up-regulation with a trend to increased the sorted cells were myeloblasts rather than typical mDC expression in the AML CR patients (p D 0.054, Fig. 3C). (Fig. 2A). Further examination of this population by flow cytome- After enrichment, CMRF-56 purity was 79.6C/¡ 3.9% and C try revealed that these cells were CD45loCD34 CD123intCD11cint, BDC purity was 28.2 p D 0.12 C/¡ 5.3% (n D 14) in AML CR confirming a myeloblast phenotype (Figure S1A). We therefore patients, which was similar to HD (n D 31, p D 0.8) BDC developed a gating strategy to aid in the elimination of blasts from purity (Fig. 3D-F). Importantly, this would generate adequate the BDC gate by including CD45, CD34 and CD304 and using mDC numbers for therapeutic vaccination. As the gross yield C strict definitions of BDC subsets (detailed in Figure S1B).

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