Abstract Purpose: The aim of this study was to establish an accelerated and de-risked workflow for the generation of potent anti-hHER2 antibody-drug conjugates (ADCs), addressing key bottlenecks that currently limit the efficiency, predictability, and overall success rate of ADC discovery. Methods: We implemented a fully integrated four-step strategy. (1) A multisite immunization protocol was developed to rapidly induce robust anti-hHER2 responses, enabling the isolation of high-titer antibodies in less than two weeks; antibodies generated with this accelerated protocol were benchmarked against a classical schedule. (2) Antibody-secreting plasma cells were isolated and ranked on the Bruker Beacon single-cell platform based on antigen binding and relative affinity prior to sequencing and recombinant expression. (3) A microscale, robotics-assisted production workflow was established to express and purify dozens of antibodies in parallel. Binding affinity was measured by bio-layer interferometry BLI, and internalization kinetics were quantified using a new lanthanide-based pH-sensitive probe. (4) A two-stage ADC synthesis pipeline was created using enzymatic conjugation and click chemistry. A first library of ADCs was generated by coupling 24 antibodies to MMAF at DAR4, followed by cytotoxicity assessment on patient-derived xenograft-derived cells (PDX-DC). The most promising hits were then reformatted into a second ADC library conjugated to multiple linker-payload combinations at diverse DARs with the same conjugation strategy. Results: The rapid immunization protocol consistently produced highly internalized anti-hHER2 antibodies, some surpassing those from classical schedule and trastuzumab. Beacon-based screening enabled efficient prioritization of clones with superior affinity. In the first ADC library, multiple candidates showed strong cytotoxic activity on PDX-DC models. Subsequent optimization through linker-payload engineering further enhanced potency for selected candidates. Conclusions: This integrated and optimized workflow accelerates ADC generation from immunization to functional readout while reducing technical risk at each stage. It enabled the rapid identification of novel anti-hHER2 antibodies with enhanced internalization and highly potent ADC derivatives. This approach is broadly applicable to future ADC discovery programs targeting diverse antigens. Citation Format: Eric Chabrol, Ph Marie-Claire Phélipot, Cécile Lemoigne, Julie Lichière, Nadjiba Marès, Alice Aymard, Alexandre Bagnolini, Sophie Mesnard, Caroline Huber, Matthieu Tassa, Ester Morgado, Jacques Fieschi. A fast and robust development process providing new highly internalized anti-hHER2 antibody-drug conjugates abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4403.
Chabrol et al. (Fri,) studied this question.