Payload diversification: a key step in the development of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) is a fast moving class of targeted biotherapeutics that currently combines the selectivity of monoclonal antibodies with the potency of a payload consisting of cytotoxic agents. For many years microtubule targeting and DNA-intercalating agents were at the forefront of ADC development. The recent approval and clinical success of trastuzumab deruxtecan (Enhertu®) and sacituzumab govitecan (Trodelvy®), two topoisomerase 1 inhibitor-based ADCs, has shown the potential of conjugating unconventional payloads with differentiated mechanisms of action. Among future developments in the ADC field, payload diversification is expected to play a key role as illustrated by a growing number of preclinical and clinical stage unconventional payload-conjugated ADCs. This review presents a comprehensive overview of validated, forgotten and newly developed payloads with different mechanisms of action.

The first ADC bearing the ferroptosis inducer RSL3 as a payload with conservation of the fragile electrophilic warhead.

The iron-dependent, non-apoptotic cell death, known as ferroptosis is an emerging strategy for the development of anticancer drugs. RSL3 was identified as an activator of ferroptosis through the inhibition of the glutathione peroxidase 4 (GPX4) which plays a crucial role in the cellular lipid oxidative stress. RSL3 is characterized by the presence of an electrophilic chloroacetyl moiety, namely warhead which covalently bonds to the catalytic and nucleophilic selenocysteine residue (Sec46) of GPX4. Like the major ferroptosis inducers, RSL3 suffers from lack of selectivity toward tumor cells. In this study, we report the first synthesis of an antibody-drug conjugate (ADC) containing RSL3 fragment and trastuzumab with the aim to deliver the agent selectively to tumors. The synthesis uses a judiciously chosen strategy to preserve the vital but fragile warhead. Full characterization of the ADC was accomplished, demonstrating the generation of a homogeneous DAR 8 conjugate. The robustness of the synthesis was successfully applied to another ADC associating the anti-CD74 mAb milatuzumab. The ADC induces ferroptotic cell death through reactive oxygen species accumulation and increases the activity of doxorubicin. The ADC associating trastuzumab and RSL3 may therefore offer potential applications in vectorized therapy alone or in combination with conventional chemotherapies.

Exatecan Antibody Drug Conjugates Based on a Hydrophilic Polysarcosine Drug-Linker Platform.

We herein report the development and evaluation of a novel HER2-targeting antibody-drug conjugate (ADC) based on the topoisomerase I inhibitor payload exatecan, using our hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform (PSARlink). In vitro and in vivo experiments were conducted in breast and gastric cancer models to characterize this original ADC and gain insight about the drug-linker structure-activity relationship. The inclusion of the PSAR hydrophobicity masking entity efficiently reduced the overall hydrophobicity of the conjugate and yielded an ADC sharing the same pharmacokinetic profile as the unconjugated antibody despite the high drug-load of the camptothecin-derived payload (drug-antibody ratio of 8). Tra-Exa-PSAR10 demonstrated strong anti-tumor activity at 1 mg/kg in an NCI-N87 xenograft model, outperforming the FDA-approved ADC DS-8201a (Enhertu), while being well tolerated in mice at a dose of 100 mg/kg. In vitro experiments showed that this exatecan-based ADC demonstrated higher bystander killing effect than DS-8201a and overcame resistance to T-DM1 (Kadcyla) in preclinical HER2+ breast and esophageal models, suggesting potential activity in heterogeneous and resistant tumors. In summary, the polysarcosine-based hydrophobicity masking approach allowsfor the generation of highly conjugated exatecan-based ADCs having excellent physicochemical properties, an improved pharmacokinetic profile, and potent in vivo anti-tumor activity.

Characterization of T-DM1-resistant breast cancer cells.

The development of targeted therapies has drastically improved the outcome of patients with different types of cancer. T-DM1 (trastuzumab-emtansine) is an antibody-drug conjugate used for the treatment of HER2-positive breast cancer combining the FDA approved mAb (monoclonal antibody) trastuzumab and the microtubule cytotoxic agent DM1 (emtansine). Despite clinical successes achieved by targeted therapies, a large number of patients develop resistance during treatment. To explore mechanisms of resistance to T-DM1, the MDA-MB-361 HER2-positive breast cancer cell line was exposed in vitro to T-DM1 in the absence or presence of ciclosporin A. Previously reported mechanisms of resistance such as trastuzumab-binding alterations, MDR1 upregulation, and SLC46A3 downregulation were not observed in these models. Despite a decrease in HER2 expression at the cell surface, both resistant cell lines remained sensitive to HER2 targeted therapies such as mAbs and tyrosine kinase inhibitors. In addition, sensitivity to DNA damaging agents and topoisomerase inhibitors were unchanged. Conversely resistance to anti-tubulin agents increased. Resistant cells displayed a decreased content of polymerized tubulin and a decreased content of ßIII tubulin although the downregulation of ßIII tubulin by siRNA in the parental cell line did not modified the sensitivity to T-DM1. Both cell lines resistant to T-DM1 also presented giant aneuploid cells. Several SLC (solute carrier) transporters were found to be differentially expressed in the resistant cells in comparison to parental cells. These results suggest that some characteristics such as increased baseline aneuploidy and altered intracellular drug trafficking might be involved in resistance to T-DM1.