Abstract 537: Preclinical development and activity of Anti-FLT3 nanoworms in acute myeloid leukemia

Abstract

Abstract Introduction: Mutations in FMS-like tyrosine kinase 3 (FLT3) occur in ~30% of acute myeloid leukemia (AML) cases causing high relapse and short survival. Acquired resistance limits the efficacy of current tyrosine kinase inhibitors against FLT3. Developing FLT3 target-specific antibodies has gained interest as a therapeutic approach for AML. Single-chain antibody fragments (scFv) are as specific as full-length monoclonal antibodies (mAbs), yet they outperform mAbs due to lower immunogenicity and a faster development process via recombinant protein engineering. However, scFvs have short circulating half-lives in humans and are difficult to purify. To overcome this, we developed anti-FLT3 scFv fused with an elastin-like polypeptide (ELP), A192, which aids in protein purifyication and extending its half life. Methods: The α-FLT3 scFv gene was fused to the amino terminus of (VPGAG)192 (A192), in the pET-25b(+) vector, purified and refolded for maximum biological activity. Binding specificity of α-FLT3-A192 was analyzed using FLT3 ITD+ AML cell lines MOLM-13 and MV4-11 and FLT3−U937 cells via flow cytometry. Viability and apoptosis assays were performed via trypan blue and Annexin-V at 72 hours, respectively. Immunoblot analyses were performed to assess the effect of α-FLT3-A192 on FLT3/STAT5/ERK signaling pathway. In vivo activity was analyzed by engrafting 106 MOLM-13 cells/ NOD scid mouse, treating with 220 uM of A192 or α-FLT3-A192 (N=8) on day 7, 10, 13 and 16, and sacrificing on day 17. Engraftment was analyzed in peripheral blood and bone marrow by HuCD45 stain and measured by flow cytometry. Kaplan Meier survival analysis was performed in MOLM-13 murine model. Results: Binding of α-FLT3-A192 (10 μM) vs. A192 (25 μM) was observed in MOLM-13 (MFI 1.0 vs 2.0; p<0.0001) and MV4-11 cells (MFI 1.0 vs 1.36; p=0.03). Contrarily, U937 cells had no binding. Cell viability decreased in MOLM-13 cells treated with α-FLT3-A192 vs. A192 at 10µM (p=0.0002, 23%), and 25µM (p<0.0001, 34%) and in MV4-11 cells at 10 µM (p<0.0001, 71%), and 25 µM (p<0.0001, 80%) vs. A192. Consistently, apoptosis increased in cells treated with 25 µM α-FLT3-A192 vs A192 (MOLM-13: p=0.007, 42.76% vs 5.27%; (MV4-11: p=0.003, 62.77% vs 25.36%). Mechanistically, α-FLT3-A192 inhibited both phospho-STAT5 and phospho-ERK in MOLM-13 and MV4-11 cells. In vivo analyses showed that spleens of mice treated with α-FLT3-A192 weighed less compared with A192 (0.48 vs 126 mg, p=0.03). Additionally, α-FLT3-A192 reduced engraftment in the peripheral blood (%huCD45- 17.5 vs 43.44, p=0.007) and bone marrow (%huCD45- 10.2 vs 26.1%, p<0.0001). Mice treated with α-FLT3-A192 had significantly longer OS than A192 group (median survival: 36 vs 30 Days, p=0.0015). Pharmacokinetic (PK) analysis showed that the terminal half-life of α-FLT3-A192 in NSG mice was ~ 14.9 hours. Conclusion: α-FLT3-A192 nanoworms present an effective FLT3 targeting strategy with high pharmacological activity and PK properties in cellular and xenograft murine models of AML. V.P.V. and M.P. contributed equally to the manuscript Citation Format: Vijaya Pooja Vaikari, Mincheol Park, John Andrew Mackay, Houda Alachkar. Preclinical development and activity of Anti-FLT3 nanoworms in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 537.