Inducible MyD88 Signaling Enhances the Anti-AML Activity of BiTE-Secreting ENG T-Cells

Background: Pediatric and adult acute myeloid Leukemia (AML) is a disease with poor prognosis due to its high relapse rate and treatment related mortality. Adoptive immunotherapy has the potential to improve outcomes and CD123 present a promising immunotherapy target for AML. CD123-specific engager (ENG) T-cells are cells genetically modified to secrete bispecific T-cell engagers (BiTEs) that recognize CD3⁺ on T-cells and CD123⁺ on AML. We had previously shown that inclusion of an inducible MyD88/CD40 costimulatory molecule, which is activated by a chemical inducer of dimerization (CID), improved anti-tumor effect against CD123+ targets in vitro. We now wanted to determine, which component of the costimulatory molecule is critical for the improved effector function of CD123-ENG T cells and perform in vivo studies.

Methods: We generated a panel of retroviral vectors encoding CD20 as a safety switch, CD123-ENG, and an inducible costimulatory molecule consisting of a myristoylation sequence, FKBP dimerizer domains and one of three signaling costimulatory domains (MyD88 (iM) ,CD40(iC) or MyD88/CD40(iMC)). A vector specific for CD19 (CD20.CD19-ENG.iMC) and non-transduced (NT) T-cells served as controls. 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 flow cytometry, ELISA and luciferase based cytotoxicity assays. We evaluated antitumor activity and persistence in vivo in a xenograft AML model.

Results: We successfully generated CD20.CD123-ENG.iM, CD20.CD123-ENG.iC and CD20.CD123-ENG.iMC T-cells. Transduction efficiency ranged from 45 to 70% (±5). The immunophenotype of CD123-ENG T-cells was predominandly CD8+ effector memory. CD20.CD123-ENG T-cells expressing inducible costimulatory molecules secreted higher levels of CD123-specific BiTEs, IL2 and IFNγ in comparison to CD20.CD123-ENG T-cells when cocultured with of MOLM-13 (CD123⁺) and CID (p<0.005, N=3). In the presence of CID, CD20.CD123-ENG.iM or CD20.CD123-ENG.iMC T-cells were able to sequentially kill AML cells for up to 5 stimulations, as opposed to CD20.CD123 ENG, CD20.CD123 ENG.iC and NT T-cells (n=3, E:T=1:1). We evaluated the in vivo anti-AML activity of ENG T cells using an AML cell line expressing firefly luciferase (MOLM-13.ffluc) to allow for serial bioluminescence imaging. MOLM-13.ffluc bearing mice were treated with one i.v. dose of effector cells (CD20.CD123-ENG, CD20.CD123-ENG iM, CD20.CD123-ENG.iC, CD20.CD123-ENG.iMC or NT T-cells ) and 2 doses/week of CID for 2 weeks (100mg/mice, i.p.). CD20.CD123-ENG.iMC and iM T-cells had potent anti-leukemic activity, resulting in a significant survival advantage in comparison to CD20.CD123-ENG T-cells group (P<0.005, n=10 animals per group).

Conclusion: We demonstrate here that inducible MyD88/CD40 costimulation significantly enhances the anti-AML activity of CD123-ENG T cells in vitro and in xenograft models. Mechanistic studies revealed that MyD88 costimulation is critical for the observed benefit. Thus, genetically modifying T-cells to express BiTEs and an inducible MyD88 costimulatory molecule may present a promising cell product for the immunotherapy of CD123+ hematological malignancies.