Extended Data Fig. 2: HER2-targeted ABCs synthesized via site-specific cysteine conjugation.

a. Non-reducing SDS-PAGE analysis of site-specifically conjugated Cy5.5-HER ABCs. Two independent experiments were performed with similar results. b. Flow cytometry histograms showing enhanced BT474 cell uptake for site-specific Cy5.5-HER2 ABC compared to BPD alone. The x-axis represents the Cy5.5 fluorescence intensity. c. and d. Quantification of cell uptake based on mean fluorescence intensity from flow cytometry (n = 3 biological replicates). e. and f. Flow cytometry histograms (The x-axis represents the Cy5.5 fluorescence intensity) and quantification, respectively, for cell uptake studies comparing site-specific Cy5.5-HER2 to stochastically functionalized lysine-based Cy5.5-HER2 ABC with BAR of 1 (BT474 cells, 20 µg/mL, 60 min incubation). Both ABCs have the same 5% Cy5.5 concentration. ABC^K is the lysine-conjugated Cy5.5-HER2 ABC prepared from commercial Trastuzumab. We note that this ABC was stored at 4 °C for ~1.5 years prior to this study, demonstrating excellent long-term storage stability. T-ABC^K is a stochastic Lys conjugate prepared using the engineered Trastuzumab designed for cysteine conjugation; this construct is designed to rule out differences in cell uptake between Lys-conjugated commercial Trastuzumab and the engineered antibody. Finally, T-ABC^C is the site-specific cysteine conjugate Cy5.5-HER2. Results are presented as mean ± SEM (n = 3 biological replicates). Statistical analysis was done using a 2-tailed t-test. For these statistical tests, NS denotes non-significant; **, P < 0.01.