Dual-Targeting CD123 and FLT3: A Promising BiTE Approach for Acute Myeloid Leukemia Therapy

A severe form of blood cancer, acute myeloid leukemia (AML), can be addressed by bispecific T cell engager (BiTE) agents that reroute T cells to target cancer cells by binding to CD3 and a tumor-associated antigen (TAA). Although BiTEs have shown effectiveness in clinical settings, resistance can arise due to the absence of a specific TAA. A hypothesis was put forth that developing a BiTE agent that targets multiple TAA might lower the chances of relapse. FLT3 and CD123 were chosen for the creation of a dual-targeting BiTE agent (dBiTE).

A half-life extended (HLE) CD123-FLT3 dBiTE agent was tested in various settings, including in vitro studies, mouse xenografts, and non-human primate (NHP) tolerability assessments. The agent exhibited high affinity in the nanomolar range for human and NHP FLT3, CD123, and CD3, and potent picomolar efficacy in cytotoxicity assays utilizing human T cells or NHP peripheral blood mononuclear cells (PBMCs). It achieved complete elimination of single-positive cells at concentrations comparable to those required for double-positive cells. In a mouse xenograft model, the agent demonstrated significant anti-tumor activity and prolonged survival beyond three weeks at various doses. In cases with single-positive tumors, the survival benefit was indeterminable due to the high survival rate, indicating the efficacy of both targeting mechanisms. In NHP studies, the agent had a half-life of 52 hours.

Post-dosing, FLT3 mRNA levels, indicative of FLT3-expressing cells, were found to decrease in the blood. However, repeated dosing led to intolerance and cytokine release, with some cytokines being reduced and others increased. It is known that CD123 is expressed on endothelial cells (ECs) and its expression is elevated under inflammatory conditions. An immunohistochemical survey revealed that CD123 is expressed on human and NHP monocytes/macrophages and ECs, with limited distribution in certain tissues.

It was hypothesized that cytokine release post-administration could lead to an increase in CD123 expression, potentially amplifying cytokine levels with repeated dosing. CD123 was found on primary human umbilical vein endothelial cells (HUVECs). In co-cultures with T cells, a CD123-specific BiTE induced CD123 expression on HUVECs at concentrations that also triggered redirected lysis, T cell activation, and cytokine secretion. To further investigate the BiTE-induced upregulation of CD123 on ECs, HUVECs were exposed to supernatants from a TDCC assay or recombinant cytokines. Both the assay supernatant and TNFα induced more than a two-fold increase in CD123 expression on HUVECs, but not on CD123-negative primary human pulmonary microvascular ECs.

These findings suggest that exposure to a CD123 mono-targeting BiTE could enhance CD123 expression on ECs, possibly through the BiTE-induced secretion of TNFα. Further research is ongoing to elucidate these mechanisms.

In conclusion, the CD123-FLT3 HLE dBiTE agent showed activity against both double-positive and single-positive target cells in vitro and in vivo. Selecting appropriate TAA for dBiTE agents is crucial for enhancing therapeutic efficacy while ensuring safety.