Thiosemicarbazones have been increasingly recognized for their potential as effective anticancer agents. 1 Specifically, those belonging to the di-2-pyridyl-thiosemicarbazone (DpT) class have shown great promise. The hallmark of DpTs is their NNS binding motif that facilitates coordination to metal ions such as Cu(II) and Fe(II). Metal-ion coordination affects various cellular targets, ultimately impacting the viability of cells by, for instance, inhibition of ribonucleotide reductase 4 , upregulation of N-myc downstream regulated gene 1 (NDRG1) , downregulation of cyclin D1 leading to cell cycle arrest , as well as others. 2,3,8 Among the many DpTs studied over the years, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) (Figure ) emerged as the most prominent candidates by exhibiting excellent selective antiproliferative activity both in vitro and in vivo against a wide variety of cancer types. 2, For example, these two compounds substantially suppress tumor growth and metastasis in murine xenograft models of pancreatic cancer and osteosarcoma. However, Dp44mT displays side effects like cardiac fibrosis and methemoglobin formation at higher doses. DpC, on the other hand, has demonstrated superior in vivo tolerability and anticancer activity compared to Dp44mT while minimizing systemic toxicity. Consequently, DpC entered phase 1 clinical trials in 2016 (NCT02688101), which concluded in 2019. Despite mitigating many of the drawbacks of Dp44mT, DpC still required treatments in precise dosing and caused pulmonary inflammation at higher doses. Therefore, the need to improve the selectivity and the therapeutic index of these molecules persists as their full potential remains untapped. A particularly effective approach to address selectivity limitations is the conjugation of drug molecules to cell-targeting antibodies. Over the years, several antibody-drug conjugates (ADCs) that combine the targeting ability of monoclonal antibodies (mAbs) with potent cytotoxic effects of drug payloads have been developed. Ideally, a mAb's targeting ability allows precise delivery of the conjugated drug to the tumor site, thereby reducing systemic side effects and broadening the therapeutic window of the drug. One of the first ADCs approved by the FDA (Trastuzumab emtansine (T-DM1)) was based on trastuzumab (Tras), a mAb that targets and binds the HER2 receptors in cancer cells. The binding is known to inhibit signaling pathways promoted by the HER2 receptor, hampering cell proliferation and survival. While Tras itself has been immensely successful in treating HER2-positive breast cancer, the use of T-DM1 has helped to combat unwanted resistance development and reduce the serious side effects of unconjugated emtansine, such as cardiac toxicity. The advent of T-DM1 proved to be pivotal in the development of ADCs as it pioneered the subsequent approval of several ADCs for different cancers. Trastuzumab inhibits downstream pathways like mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3 kinase (PI3K)/AKT, making cancer cells susceptible to cell death-inducing agents like the DpTs. A priori, we hypothesized that conjugating DpTs to Tras could display synergistic effects, considering that the DpTs upon iron chelation are also known to affect similar cellular pathways with respect to Tras. Here, we employed strain-promoted oxidation-controlled cyclooctyne-1,2-quinone (SPOCQ) cycloaddition technology and strain-promoted azide-alkyne click reaction (SPAAC) for the antibody modification and conjugation of DpTs to Tras. Further, we explored the effects of conjugating the mAb to different positions of the DpT structural motif on the antiproliferative activity of the synthesized ADCs.