FIGURE 2.

Directed evolution of CD47 using classical library creation strategies. A, CD47 library designs. Left, crystal structure of SIRPα (green) and CD47 (magenta) is shown in cartoon representation. Amino acid positions randomized in the libraries are highlighted and shown in sphere representation. CD47 is divided into three different patches as follows: core (cyan), contact region 1 (gray), and contact region 2 (orange). Randomizing combinations of these three patches created multiple CD47 libraries that were selected independently for the first three rounds but mixed to select as a single library on the last round of selection. Middle right, tables of randomized positions with possible amino acid variations and the corresponding degenerate DNA codons (noted in the parentheses). a.a., amino acid. B, schematics of the criteria at each round of CD47 library selections. MACS, magnetic activated cell sorting; FACS, fluorescence activated cell sorting; and SA, streptavidin. C, summary of sequences of selected CD47 variants. The position of mutated residues and their corresponding sequence in WT is denoted at the top of the table. D, binding titration curves of SIRPα a2d1 on yeast expressing WT CD47 (black) or selected variant A9 (yellow), B4 (blue), or C1 (green). Cells were stained with serial dilutions of biotinylated SIRPα a2d1 and analyzed by flow cytometry. Data represent raw (top) or normalized (bottom) mean fluorescence intensity ± S.E. Data are representative of two independent experiments. a.u., arbitrary units.