FIGURE 3.
Targeted residues in Fc region and screening process of Fc libraries. A, amino acid sequence of a human IgG1 Fc polypeptide to be targeted for the construction of Fc libraries. The amino acid sequence of a human IgG1 Fc region, starting from the hinge region and ending with the carboxyl terminus of the CH3 domain, is shown in single letter notation and is numbered according to the EU system of Edelman et al. (35). The amino acids underlined and in boldface type were randomized in constructing the libraries as described under “Experimental Procedures.” Below each of these amino acids is a 1, a 2, or a 3, which indicates that DNAs encoding variants at the corresponding site were included in a Tier 1, 2, or 3 library as described under “Experimental Procedures.” B, diagram to show the primary screening and initial combinatorial screening for substitutions that enhance binding to FcγRIIIA. The rectangle labeled SIG represents a polynucleotide encoding a signal sequence, which facilitates protein secretion from mammalian cells. A region encoding a hinge region is represented by a horizontal line labeled hinge. A rectangle labeled Fc polypeptide represents a polynucleotide encoding an Fc polypeptide chain. The five-pointed and four-pointed stars mean that the polynucleotides encoding the Fc polypeptide chains contain randomized codons at selected positions as explained under “Experimental Procedures.” The circles labeled with VH and VL represent the regions encoding a heavy chain variable region and a light chain variable region, respectively. The + + and − − in the rectangles labeled Fc polypeptide mean that these regions include mutations such that the encoded Fc polypeptide chain will have the substitutions E356K + D399K and K392D + K409D, respectively.