Fig. 1. TXC conjugation strategy and biophysical characterization. (A) Iodoacetamide-modified payloads are conjugated to engineered cysteines on XTEN. The XTEN-payload intermediate is reacted with SMCC to install a maleimide at the N-terminus. Finally, mal-XTEN-payloadn is conjugated to the two engineered Cys of a THIOMAB antibody to give the final THIOMAB antibody/XTEN-payloadn conjugate (TXC). (B) Structure of αHer2/XTEN-May16 TXC (average DAR = 16); (C) HPLC chromatogram of purified mal-XTEN-Mayn intermediate generated via initial conjugation methods, giving a heterogeneous product with an average n = 8 (red trace) and optimized conjugation methods giving a more homogeneous product with n = 9 (blue trace); (D) analytical size-exclusion chromatogram (SEC) of globular protein standards, XTEN standards of different nominal molecular weights indicated in kDa, and αHer2/XTEN-May16; (E) IC-IEF fingerprint for αHer2/XTEN-May16 (blue) from which an approximate pI value of 4.1–5.6 was derived. Unconjugated antibody spiked with pI markers (red) and blank (black) are shown for reference.
