Fig. 5.

Fab dimers can be trapped with engineered disulfides. (a) Representative SEC chromatograms of select cysteine variant and WT anti-Her2 Fabs after affinity chromatography. Peaks are labeled as monomer (M), dimer (D), and higher-order (HO). (b) Summary of monomer (green diamonds), dimer (blue circles), and higher order (red squares) species for all cysteine variants by SEC post-affinity chromatography. Data represent percentage of species based on integration of SEC peaks. Multiple symbols for a given cluster represent multiple designed cysteine variants. The identities of all variants and associated numeric SEC data are provided in Supplementary Tables S3-S5. (c) Correlation between expression and in vitro coupling data for disulfide-trapped dimers. (d) Summary of data from in vitro coupling of selected cysteine variant Fabs. The data reflect the % monomer (green diamonds), dimer (blue circles), and higher-order (red squares) species after assembly for the designed and mismatched (MM) variants run as negative controls. For MM variants the pairings of HCs and LCs with single cysteine mutations were scrambled in four separate combinations. The identities of all variants and associated numeric data are provided in Supplementary Table S6. (e) OX40 receptor activation by variant and WT Fab and full-length IgG antibodies based on an NFκB luciferase reporter assay. VH-16 represents Fab cysteine variant VH(S113C) / CH1(G178C) and CH1-176 represents Fab cysteine variant VH(P14C) / CL(D151C). Fold receptor activation represents normalized RLU relative to cells alone. For the IgG1 versions, +XL and -XL indicate the presence or absence of secondary crosslinker antibody, respectively. RGY represents a triple Fc variant that promotes IgG hexamer formation. No crosslinker was used for the RGY version or the Fab monomer or dimer versions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)