Fig. 1

DSP107 structure and binding characteristics A) 3D schematic representation of trimeric DSP107 comprising genetically fused ECDs of human SIRPα (blue) to 4-1BBL (green). 3D structures are from Protein Data Bank and the 3D model was obtained based on PDB structures (4-1BBL PDB IDs: 2X29, 6FIB, 6BWV, 6CPR, 6D3N, 6CU0, 6MGE, 6MGP, 6A3V; SIRPα PDB IDs: 2JJS, 2JJT, 2UV3, 2WNG, 4CMM, 4KJY, 6BIT. B) SEC-MALS analysis of purified DSP107 showing the protein forms into a trimer in solution. Calculated molar mass is 210 g/mol/ 190 kDa C) Binding of DSP107 (red) or a DSP-MOCK (purple), to CD47:Fc immobilized on the plate and measured by BLI. no binding is observed to a chip without CD47:Fc (blue) D) Binding of DSP107 (red) or a DSP-MOCK (purple) to 4-1BB:Fc immobilized on the chip or without 4-1BB:Fc as in C. E) Binding of DSP107-FITC to SUDHL10 cells (red). DSP107 binding was blocked by CD47 mAb (green), unstained cells (black) F) Binding of DSP107-FITC to HT1080.wt cells (red), was completely blocked by CD47 mAb (green) and not affected by s4-1BBL (blue). G) Binding of DSP107-FITC to HT1080.4-1BB cells (red), was partially blocked by CD47 mAb (blue) and completely blocked by both CD47 mAb and soluble 4-1BBL (green). H) Doublet formation of CFSE stained CHO.CD47 and CytoLightRed stained HT1080.4-1BB cells without DSP107 (top) or with 5 μg/ml of DSP107 (bottom). Doublets are detected as a double-positive FITC-APC signal (bottom, top right corner). I) Doublet formation with DSP107, DSP-MOCK and after blocking with CD47 mAb or soluble 4-1BBL, parametric paired t-test was used with respect to control sample versus treatment with DSP107/mock-DSP or blocking agnets, * p < 0.05; J) DSP107 binding to a mixture of PBMCs (blue), SUDHL4 cells (purple) and RBCs (red) determined by flow cytometry, K) Binding of CD47 mAb to the mixture as in J. Error bars present in the figures represent SD