Myd88/CD40 Costimulation to Enhance the Antitumor Activity of CD123-ENG T-Cells for the Immunotherapy of AML

Abstract

Background: Acute myeloid leukemia (AML) represents a treatment challenge due to its poor prognosis and high associated morbidity and mortality rates. Thus, new therapies are needed. CD123-ENG T-cells are cells genetically modified to secrete bispecific antibodies that recognize CD3 (T-cells) and CD123 (AML). Preclinical studies show that these cells recruit bystander T-cells to kill CD123+ blasts in vitro and in vivo. However, CD123-ENGs are unable to maintain sequential killing capability of CD123+ targets. To overcome this limitation, we have devised an approach that provides inducible Myd88/CD40 costimulation activated by a chemical inducer of dimerization (CID). This strategy should increase the persistence, expansion and anti-AML activity of CD123-ENG T-cells.Methods: We generated a retroviral vector encoding a CD20 safety switch, CD123-ENG, and the inducible costimulatory molecule MyD88.CD40 (iMC) linked by 2A sequences (CD20.2A.CD123-ENG.2A.iMC). We used a vector specific for CD19 (CD20.2A.CD19-ENG.2A.iMC) and non-transduced (NT) cells as controls for non-specific effects of the iMC construct. We genetically modified T-cells using a retroviral transduction protocol and evaluated their effector function +/- CID. We assessed the effector function of transduced T-cells using standard immunological assays and a flow cytometry based sequential killing assay.Results: We successfully generated CD20.CD123-ENG.iMC T-cells, which maintained a transduction efficiency above 50% throughout our study period. We performed coculture and cytotoxicity assays using NT, CD20.CD123-ENG, CD20.CD19-ENG.iMC and CD20.CD123-ENG.iMC T-cells +/-CID as effectors and MOLM13 (CD123+), Kg1a (CD123+) and K562 (CD123-) as targets. Cocultures were performed +/- CID. CD20.CD123-ENG.iMC T-cells maintained CD123 antigen specificity, as evidenced by cytotoxicity and cytokine assays. CD20.CD123-ENG.iMC T-cells + CID secreted increased IL-2 and IFN-γ in the presence of CD123+ targets (KG1a and MOLM13) when compared to baseline and to CD20.CD123-ENG T-cells. In addition, CD20.CD123-ENG.iMC T-cells + CID displayed enhanced sequential killing capabilities at a 1:1 ratio, compared to CD20.CD123-ENG T-cells.Conclusion: CD20.CD123-ENG.iMC T-cells recognize and kill CD123+ AML blasts in an antigen dependent manner. In addition, control ENG.iMC T-cells have no antitumor activity, indicating that activation of Myd88/CD40 does not induce nonspecific AML blast killing. CD20.CD123-ENG.iMC T-cells have improved effector function in the presence of CID as judged by increased cytokine production and their ability to sequentially kill CD123+ target cells in vitro. Based on our promising in vitro experiments, we have initiated in vivo studies in AML xenograft models, which are currently in progress. DisclosuresLeen:Marker: Equity Ownership.