Trastuzumab binds HER2 extracellular subdomain IV (membrane-proximal), blocking ligand-independent HER2 signaling and enabling Fc-mediated ADCC. Pertuzumab binds subdomain II (dimerization arm), directly blocking HER2/HER3 heterodimerization. The two epitopes are non-overlapping, which is why Trastuzumab + Pertuzumab combination therapy is synergistic. For epitope mapping of novel anti-HER2 candidates, individual HER2 subdomain proteins can be used to determine which domain is targeted.

Both are HER2-targeting ADCs but differ in key design parameters. T-DM1 (Kadcyla) uses a non-cleavable linker and the maytansinoid payload DM1, with a drug-antibody ratio (DAR) of ~3.5. T-DXd (Enhertu) uses a cleavable linker and the topoisomerase I inhibitor DXd, with a higher DAR of ~8 and a membrane-permeable payload that enables a bystander killing effect on neighboring HER2-low cells. This bystander effect is why T-DXd is effective in HER2-low tumors (IHC 1+ or 2+/FISH-negative) where T-DM1 is not.

HER2 IHC scoring uses a 0-3+ scale: 0 (no staining), 1+ (faint, partial membrane staining in >10% of cells), 2+ (weak-to-moderate complete membrane staining in >10% of cells — equivocal, requires FISH confirmation), 3+ (strong complete membrane staining in >10% of cells — HER2-positive). With the approval of T-DXd for HER2-low tumors, the clinical relevance of IHC 1+ and 2+/FISH- categories has increased significantly.

Soluble HER2 (sHER2/ECD) is the ~110 kDa ectodomain shed from the tumor cell surface by ADAM metalloprotease cleavage. Elevated serum sHER2 (>15 ng/mL) correlates with HER2 amplification, tumor burden, and poor prognosis. sHER2 can also act as an antigen sink — sequestering Trastuzumab in the bloodstream before it reaches the tumor. Our HER2 ELISA kit quantifies sHER2 in serum for biomarker and pharmacodynamic studies.

Use SKBR3 or BT-474 cells (HER2 3+ target cells) co-cultured with peripheral blood NK cells or an engineered ADCC reporter cell line (e.g., Jurkat-FcγRIIIa-NFAT-Luc). Add serial dilutions of Trastuzumab biosimilar vs reference standard. Measure target cell killing (LDH release or calcein-AM release for primary NK cells) or reporter luminescence (for reporter cells) after 4-6 hours. The dose-response curves should be parallel with a relative potency within 80-120% of the reference.