As such, the extent to which the clinical activity of these second-generation HER2-targeting ADCs will be limited by drug resistance remains to be seen. conclude that the T-DM1 resistance mechanisms most strongly supported by the experimental data relate to dysfunctional intracellular metabolism of the construct and subversion of DM1-mediated cell killing. Loss of dependence on signalling initiated by HER2CHER2 homodimers is not substantiated as a resistance mechanism by clinical or experimental studies, and the impact of EGFR expression and tumour immunological status requires further investigation. These findings are instructive with respect to strategies that might overcome T-DM1 resistance, including the use of second-generation anti-HER2 antibodyCdrug conjugates that deploy alternative linker-payload chemistries. mRNA and HER2 protein expression, which might have contributed to reduced event-free survival in the T-DM1 treatment arm.7 However, the fact that pCR was achieved in 44% of patients who received T-DM1 plus pertuzumab in KRISTINE and the Phase 2 PREDIX trial8 without the use of potentially toxic systemic chemotherapy is notable, indicating that a subset of patients, particularly WP1130 (Degrasyn) those with high and homogeneous HER expression, might not require neoadjuvant chemotherapy, and that their treatment could be de-escalated.9 Other authors have considered why T-DM1 plus pertuzumab does not provide greater efficacy than pertuzumab and trastuzumab in combination with chemotherapy in the metastatic and neoadjuvant settings, proposing tumour heterogeneity, clonal selection, bystander effects and receptor downregulation by competitive binding as potential biological explanations.10 The KATHERINE trial An interim analysis of the ongoing KATHERINE trial in patients with HER2-positive early breast cancer with residual invasive disease in the breast or axilla after neoadjuvant treatment with trastuzumab and a taxane revealed that use of adjuvant T-DM1 conferred a lower risk of recurrence of invasive breast cancer than adjuvant trastuzumab, although with a higher rate of adverse events.11 On the basis of these results, T-DM1 recently received regulatory approval for this neoadjuvant indication.12 Mechanisms of action of T-DM1 T-DM1 has multiple mechanisms of action, from the selective delivery of DM1 to HER2-positive tumour cells through to trastuzumab-mediated inhibition of HER2 signalling, inhibition of HER2 extracellular domain shedding and induction of antibody-dependent cell-mediated cytotoxicity (ADCC) (Fig.?2). Open in a separate window Fig. 2 Mechanisms of action of T-DM1.T-DM1 exerts anti-tumour activity via at least three distinct mechanisms. As for trastuzumab, engagement of HER2 receptors by T-DM1 inhibits downstream signalling pathways (via RASCmitogen-activated protein kinase [MAPK] and phosphatidylinositol 3-kinase [PI3K]CAKTCmammalian target of rapamycin [mTOR]) and ectodomain shedding while also eliciting immune effector cell function (e.g. antibody-dependent cellular cytotoxicity) mediated via Fc receptors. T-DM1CHER2 complexes are also internalised via receptor-mediated endocytosis, after which endocytic vesicles mature through the endosomal pathway for ultimate delivery to lysosomes. Trastuzumab is proteolytically degraded in lysosomes, liberating lysine-MCC-DM1 for active transport into the cytoplasm, where it inhibits tubulin polymerisation resulting in failure of the mitotic spindle and ultimate mitotic catastrophe. Tumour-selective delivery and cytotoxicity of DM1 The tumour selectivity of T-DM1 is conferred by the exquisite specificity of trastuzumab to subdomain IV of the HER2 receptor on the surface of antigen-positive cells. Following binding to this extracellular epitope, the ADCCreceptor complex is internalised into early endosomes by receptor-mediated endocytosis. The endocytic vesicles either mature and fuse with the lysosome or are recycled to transport the ADCCreceptor complex back to the plasma membrane.13 Lysosomal degradation of the antibody component of T-DM1 results in the liberation of lysine-MCC-DM1, which, as a lysine derivative, is positively charged at physiological pH and therefore membrane-impermeable.14 As such, lysine-MCC-DM1 requires active transport to WP1130 (Degrasyn) efficiently cross the lysosomal membrane before it can engage its molecular target in the cytoplasm and also has a limited APC ability to diffuse to proximal, antigen-negative cells to induce a cytotoxic bystander effect.15 Proteolysis of trastuzumab spares the MCC moiety on DM1, but this retained linker does not WP1130 (Degrasyn) impair payload potency, ensuring that lysine-MCC-DM1 liberated from lysosomes binds efficiently to tubulin to prevent microtubule polymerisation.1,16 Analogous to vinca alkaloid chemotherapeutics, microtubule depolymerisation by DM1 prevents the assembly of a functional mitotic spindle, resulting in unattached kinetochores, cell-cycle arrest in metaphase with presentation.