ALBERT

Description: 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.

Description: Acute myeloid leukemia (AML) is a blood cancer arising from clonal proliferation of myeloid progenitors harboring oncogenic mutations. AML patients have poor clinical prognosis and limited therapeutic options, with myeloablation followed by hematopoietic stem cell transplantation (HSCT) as the only curative treatment. The conditioning regimens used indiscriminately kill all highly proliferative cell types, leading to life threatening side effects, and are also potentially ineffective against quiescent AML subpopulations. To address the challenges of efficacy and resistance in targeting AML, we developed a humanized bispecific antibody (CDX) that recruits T cells through CD3 to target and kill Fms-like Tyrosine Kinase 3 (FLT3) expressing cells. Normal FLT3 expression is mostly restricted to hematopoietic stem and progenitor cells (HSPCs) and its activation, through binding of FLT3 ligand (FLT3L), promotes normal differentiation of downstream blood lineages. FLT3 is also expressed on AML blasts in a majority of patients and is thought to promote survival and proliferation of AML. Although multiple promising tyrosine kinase inhibitors (TKIs) have been and are being developed to specifically target FLT3, secondary mutations leading to resistance against FLT3-TKIs remain a major obstacle. Surface expression of FLT3 in leukemic cells, as well as in HSCs, makes it an excellent target for T cell mediated conditioning that specifically eliminates both AML blasts and HSPCs, allowing for subsequent HSCT. In addition, HDX mass spectrometry revealed that our CDX binds an extracellular, membrane proximal FLT3 domain, which does not compete with FLT3L and is outside the regions commonly mutated in AML, therefore, unlike other therapies, targets all FLT3-expressing cells, regardless of mutations in the receptor. We found that in vitro treatment with CDX redirected primary human T cells to kill multiple AML-derived FLT3+ cell lines, some harboring common FLT3 mutations, at EC50 ranging from 0.3 pM to 15 pM. Additionally, the presence of FLT3L (10 nM) had no effect on the EC50 of CDX binding to FLT3+ target cells. To test CDX in vivo, we generated xenograft mice engrafted with primary human peripheral blood mononuclear cells (PBMCs) and MV4;11 cells (FLT3+ AML cell line), which were engineered to stably express EGFP. Mice were treated intravenously with 3 doses of CDX at a concentration of 0.1 mg/kg or left untreated over 1 week (n=5). Progression of AML was tracked by monitoring the frequency of EGFP-MV4;11 cells in peripheral blood (PB) of xenografted mice, which were euthanized if they presented with cachexia, hind-leg paralysis, and severe weight loss (symptoms of AML progression). Treatment with CDX significantly delayed the proliferation of MV4;11 cells, based on lower PB frequencies of EGFP-MV4;11, and increased median survival by 28 days relative to untreated mice. To more stringently model CDX efficacy in a clinical setting, we injected EGFP-MV4;11 cells into humanized mice that were generated by transplantation of human cord blood derived, CD34+ HSPCs and displayed stable multi-lineage engraftment (~20% human CD45+) with significant populations of T cells at 35 weeks. We observed that T cells in CDX-treated mice over-expressed the immune checkpoint receptor PD1, relative to untreated mice, and hypothesized that PD1 inhibition could enhance MV4;11 clearance via CDX treatment by preventing T cell exhaustion. Treatment regimens consisted of 3 doses of CDX (0.1 mg/kg), anti-PD1 antibody (100 μg), or a combination of CDX and anti-PD1 (n=5), followed by transplant with autologous mouse bone marrow cells. Treatment with CDX alone led to decreased PB frequencies of EGFP-MV4;11 cells, but only modestly improved median survival relative to anti-PD1 alone (9 days). In contrast, co-treatment with CDX and anti-PD1 antibody significantly increased median survival (16 days) and was associated with decreased PD1 expression on T cells. In addition, administration of CDX alone or in combination with PD1 in humanized mice resulted in efficient elimination of the human hematopoietic compartment from the mouse bone marrow. Based on our findings, CDX shows promise for treatment of AML with concurrent conditioning for HSCT with improved specificity compared to standard of care and even displays synergy with checkpoint inhibition of PD1. Disclosures Sirochinsky: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Shrestha: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.