Preclinical Development of Anti-FLT3 CAR-T Therapy for the Treatment of Acute Myeloid Leukemia
Authors: Shrestha Elina , Liang Raymond , Sirochinsky Carina , Ben Jehuda Ronen , Sandler Vladislav 
Published: November 2020

FMS-like tyrosine kinase 3 (FLT3) is a class III transmembrane receptor tyrosine kinase involved in survival, proliferation, and differentiation of hematopoietic stem/progenitor cells. It is preferentially expressed on the leukemic cells of myeloid lineage including acute myeloid leukemia (AML) and is mutated in approximately one-third of patients with AML, resulting in constitutive signaling associated with poor disease prognosis. Although small molecule inhibitors targeting FLT3 have shown some success in clinical trials, they only work transiently while resistance develops in virtually all patients. The only proven curative treatment for the relapsed or refractory (R/R) AML is allogenic hematopoietic stem cell transplantation (HSCT) which requires highly toxic conditioning regimens often associated with fatal side effects. Thus, there still remains an urgent need for the development of safe yet effective new therapies for the treatment of AML.

We developed a novel chimeric antigen receptor modified T (CAR-T) cell therapy targeting FLT3 to eliminate FLT3+ R/R AML leukemia via cytotoxic T lymphocytes (CTL)-mediated cytolysis. Since FLT3 is also expressed on hematopoietic stem cells (HSCs) as well as on early hematopoietic progenitors (HPs), we evaluated the conditioning efficacy of our anti-FLT3 CAR-T in addition to its anti-leukemic activity.

We first discovered a novel mouse monoclonal antibody that binds to the extracellular domain of human FLT3 with high affinity (0.8 nM EC50 in FLT3+ leukemic cell line REH) while not competing with FLT3 ligand in order to achieve unobstructed and efficient binding to FLT3. We next generated humanized single-chain variable fragment (scFv) antibodies and characterized their binding affinities. The scFv clone that exhibited highest binding to FLT3 (3.42 nM EC50 in REH cells) was used to design a third generation CAR construct with CD28 and 4-1BB costimulatory and CD3ζ activation domains. T cells isolated from peripheral blood (PB) were transduced with a lentiviral vector encoding the FLT3-CAR. Transduced cells exhibited stable expression of CAR protein and expanded over 120-fold after 18 days in culture. We demonstrated high cytotoxicity of FLT3-CAR-T cells towards AML-derived cell lines in co-culture experiments, even at effector-to-target cells ratios as low as 1:10. In vivo functionality of FLT3-CART was determined by flow cytometry analysis of leukemia burden in the peripheral blood of mice engrafted with GFP+ MOLM-13 (FLT3+ AML cell line) and treated with two doses of 4x106 control or FLT3-CAR-T cells. Compared to control, the appearance of MOLM-13 cells in peripheral blood was significantly delayed in FLT3-CAR-T treated mice. AML progression in mice was also assessed by detection of physical symptoms such as cachexia and hind-leg paralysis in terminal stages. FLT3-CAR-T treatment extended the median survival to 47 days compared to 24 days in control.

Moreover, to test if our CAR-T therapy can also efficiently eliminate FLT3+ HSCs and HPs, humanized mice generated by engrafting human cord blood CD34+ cells were injected with autologous control or FLT3-CAR-T cells. Analysis of bone marrow 18 days post treatment, showed that mice that received FLT3-CAR-T cells exhibited dramatically lower frequencies (by 57% in CD38+ and 86% in CD38-) of human CD34+ hematopoietic stem and progenitor cells than control mice, suggesting the potential of CAR-T therapy for HSCT conditioning. In conclusion, our CAR-T therapy shows robust cytolytic activity against FLT3+ cells, demonstrates high efficacy in eradicating FLT3+ R/R AML leukemia in vivo and enables bone marrow conditioning for potentially curative HSCTs by specifically targeting FLT3+ HSCs and early HPs. To prevent the potentially harmful side effects associated with CAR-T therapies, such as cytokine release syndrome and cytotoxicity towards newly transplanted HSCs post conditioning, we are currently testing FLT3-CAR-T cells equipped with inducible caspase9 or EGFRT expression based safety switch to specifically eliminate CAR-Ts by administering FDA-approved small molecules or biologics.

Disclosures

Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Sirochinsky:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.