A Multiplex Protein Microarray Platform for Preclinical Tissue Binding and Cross-Reactivity of Therapeutic Antibodies

The NexAb multiplex protein microarray platform, comprising a 7,015-spot tissue lysate array and antigen-oriented protein arrays, was developed for preclinical tissue-binding and cross-reactivity assessment of therapeutic antibodies. Four anti-HER2 antibodies, trastuzumab, pertuzumab, trastuzumab emtansine (T-DM1), and trastuzumab deruxtecan (T-DXd), were profiled with integrated ERBB2 mRNA data. On antigen-oriented arrays, cell-synthesized HER2 bound trastuzumab and pertuzumab strongly and epitope-dependently, whereas wheat-germ HER2 showed no binding. On the tissue lysate array, trastuzumab and T-DM1 bound HER2-positive cancer cell lines and tumor tissues with nearly identical profiles (correlation coefficient r = 0.991), and T-DXd bound ERBB2-low cancer spots and 38.8% of normal human tissue spots, a profile not conserved in cynomolgus monkeys. Trastuzumab, T-DM1, and pertuzumab showed no cross-reactivity with cynomolgus monkey tissues, whereas T-DXd bound 3.1% of monkey tissue spots. NexAb enables quantitative cross-reactivity profiling relevant to preclinical safety evaluation.