Figure 2 from FLT3/CD99 Bispecific Antibody–Based Nanoparticles for Acute Myeloid Leukemia

Construction and biophysical characterization of an ELP fusion protein targeting both CD99 and FLT3 receptors. A, A single-chain variable fragment targeting either CD99 receptor or FLT3 receptor was genetically fused to the amino-terminus of a high molecular weight ELP, A192. These CD99-A192 and FLT3-A192 nanoparticles were expressed in E. coli. Prior to protein refolding, CD99-A192 and FLT3-A192 were co-assembled to generate the dual-targeting CD99-A192/FLT3-A192 nanoparticles. B, Following purification, ELPs were obtained with high purity as indicated by an SDS-PAGE gel stained with Coomassie brilliant blue. On the right image, the fluorescent image of the SDS-PAGE gel shows efficient fluorescent labeling the proteins with NHS-rhodamine or FITC. C, The high purity of the ELPs was further confirmed by MALDI-TOF MS. The mass spectrum of Co-Assembled shows two sets of distinct m/z peaks corresponding to the presence of both CD99-A192 and FLT3-A192. D, The hydrodynamic radius (Rh) of the scFv-ELPs was measured using DLS at 37°C. Compared to CD99-A192 and FLT3-A192, Co-Assembled increased the Rh of nanoparticle formation. E, SEC-MALS was used to measure the absolute molar mass and radius of gyration (Rg) of the scFv-ELP nanoparticles. For all three fusion proteins, the monomers oligomerized to form nanoparticles with an absolute molar mass of 10⁷ to 5 × 10⁷ Da. F–H, The morphology of the (F) CD99-A192, (G) FLT3-A192, and (H) Co-Assembled nanoparticles was observed via negative-staining TEM, which showed an elongated, rod-like nanostructures. Scale bar: 0.1 μm. I, The transition temperatures (Tt) of the ELPs were measured at 0.4, 2, 10, and 50 μmol/L. Consistent with previous findings, the fusion of scFv to A192 lowers the transition temperature of A192; however, all formulations are expected to remain soluble at physiological temperatures.