Cytokine Induced Killer (CIK) Cells Transduced with an Anti-CD19 Chimeric Receptor Containing 4-1BB Acquire Powerful Anti-Leukemic Activity.
Authors: Marin Virna , D’Amico Giovanna , Kakuda Harumi , Dander Erica , Campana Dario , Biondi Andrea 
Published: October 2019

Abstract
CIK cells are ex-vivo expanded cells with MHC-unrestricted cytotoxicity against several tumors, except B-lineage Acute Lymphoblastic Leukemia (ALL). Transduction of an anti-CD19-ζ chimeric receptor (cTCR) in CIK cells renders them efficient killers of ALL cells. However, in order to be fully effective for ALL immunotherapy, CIK cells should be able to exert a prolonged anti-leukemic activity after infusion, proliferating and secreting cytokines which amplify the anti-tumoral immune response following ALL encounter. Since it has been shown that incorporation of costimulatory molecules into cTCRs markedly enhances target-cell stimulated proliferation and cytotoxicity in T lymphocytes and NK cells, our aim was to identify costimulatory molecules able to increase the anti-leukemic properties of anti-CD19-ζ cTCR transduced CIK cells. CIK cells were efficiently transduced with the retroviral vectors carrying different types of cTCRs: anti-CD19-ζ, anti-CD19-DAP10, anti-CD19-4-1BB-ζ and anti-CD19-CD28-ζ (average expression of GFP and cTCR, 55% for all vectors tested; n=5 each). CIK cells expressing anti-CD19-ζ, anti-CD19-DAP10, anti-CD19-4-1BB-ζ and anti-CD19-CD28-ζ cTCRs were all strongly cytotoxic against OP-1 cells after 4 hours of incubation (>60% of lysis at E: T ratio 2:1 for all receptors tested). The benefit of adding the costimulatory molecules 4-1BB or CD28 to the cTCR was evident in long-term co-coltures (6days) on a mesenchimal cells layer, with low percentages of CIK cells (E:T ratio 0.01:1). In these assays, CIK cells expressing anti-CD19-4-1BB-ζ or anti-CD19-CD28-ζ cTCRs had more potent cytotoxicity than cells expressing the anti-CD19-ζ cTCR: average cell killing was 93% (range, 89–98%), 93% (87–97%), and 14% (3–24%), respectively (n=4, each; p=0.001). By contrast, addition of DAP10 to the cTCR did not improve cytotoxicity (average cell killing, 2%). Notably, CIK cells transduced with the anti-CD19-4-1BB-ζ cTCR had higher proliferative capacity in cocultures with irradiated OP1 cells and low dose IL-2. The average fold increase after 2 weeks of colture was 2.6 (range, 2.4–3.0) for these cells, while expansion of cells transduced with anti-CD19-ζ or anti-CD19-CD28-ζ was 1.4 (1.3–1.5) and 1.7 (1.2–2.7), respectively (p=0.01). Moreover, inclusion of 4-1BB or CD28 in the cTCR caused a significant increase in cytokines release (Multiplex Flow Cytomix assay) from transduced CIK cells after 48h stimulation with irradiated OP-1 cells: in 3 different experiments, CIK cells expressing anti-CD19-4-1BB-ζ or anti-CD19-CD28-ζ cTCR, secreted respectively, 2- and 3-fold more IL-2 (2040 and 2980 pg/ml, p=0.05), 3- and 4-fold more TNF-α (2000 and 2555 pg/ml, p=0.05), 4- and 3-fold more TNF-β (813 and 578 pg/ml, p=0.005), 4- and 3-fold more IL-6 (325 and 230 pg/ml, p=0.05), 3- and 4-fold more IL-8 (1962 and 2480 pg/ml, p=0.05) compared to cells expressing anti-CD19-ζ cTCR. However, OP-1 stimulation of anti-CD19-CD28-ζ cTCR transduced CIK cells resulted in the release of significant higher amount of the immunosuppressive cytokine IL-10 than CIK cells expressing the anti-CD19-4-1BB-ζ cTCR (880 and 85 pg/ml, p=0.05). In conclusion, our results suggest that anti-CD19-4-1BB-ζ cTCR represents the best tool to improve anti-leukemic activity of CIK cells for ALL immunotherapy, as it strenghtens their cytotoxicity against leukemic cells and notably, it induces transduced cell proliferation and secretion of immuno-stimulatory cytokines.