Abstract 4054: Development of a new site-specific sulfomaleimide rebridging platform for the generation of homogeneous, stable, potent and safe antibody drug conjugates
Authors: Haeuw Jean-Francois , Marion Frederic , Dreyfus Cyrille , Akla Barbara , Zanna Laurence , Janin Marie-Claire , Beau-Larvor Charlotte , Millet Marion , Farrie Martine , Batton Alain , Thuilliez Celine , Beck Alain , Cianferani Sarah , Clerc Thierry , Maillos Philippe , Corvaia Nathalie 
Published: September 2020

Abstract
There are currently 5 antibody drug conjugates (ADC) on the market and over 80 other ADCs evaluated in the clinic. The major drawbacks of the commonly employed bioconjugation methods based on nucleophilic coupling to lysine and cysteine are the non-selectivity and the in vivo instability. These limitations may be overcome by using alternative bioconjugation chemistries. To avoid the need for antibody engineering, disulfide-bridging linkers have recently emerged for the generation of homogeneous and stable ADCs.
Here, we report a novel maleimide-based linker for site-selective bioconjugation of drugs via covalent re-bridging of reduced interchain disulfide bonds on native IgG1 antibodies. After optimization of the maleimide and linker parts, as well as of the coupling process, the sulfomaleimide re-bridging (SMR) platform was used to conjugate tubulin and DNA-targeting cytotoxic agents to model antibodies. Compared to conventional maleimide (MC)-ADCs, the SMR-ADCs were highly homogeneous by HIC-HPLC and LC-MS analysis, with DAR 4 species as the major product, and highly stable in vitro, with no deconjugation observed after incubation in different types of serum. Then, IGF-1R-targeting SMR-ADCs were synthesized with Auristatin and PNU-159682 derivatives and evaluated in in vitro and in vivo models. These ADCs exhibited potent cytotoxicity and anti-tumoral activity in 2+ and 3+ IGF-1R-expressing models at different doses (0.3 to 3 mg/kg) and schedules of administration (Q1W to Q3W, i.v.). Significant differences in in vivo efficacy were observed between SMR- and MC-ADCS constructed with Auristatin derivatives. While they were comparable in a 3+ xenograft model (MCF-7), complete regressions were indeed obtained in the 2+ CaOV3 and NCI-H2122 models after single injections of the Auristatin-based SMR-ADC, whereas the corresponding MC-ADC induced only partial responses in the same conditions. To compare their in vivo stability and tolerability, these ADCs were injected in mice at doses of 3-30 mg/kg (Q2W, i.v.). No mortality and clinical signs were observed. Body weight evolution was not affected whatever the treatment. From a histopathological perspective, no difference between both constructs could be highlighted at this point. Toxicokinetic analyses carried out from satellite animals demonstrated that there was no major difference between both ADCs and that animal exposure increased proportionally with the dose. Nevertheless, serum exposure to the payload was 2-to-7-fold higher for MC-ADC than for SMR-ADC.
Our results demonstrate that the SMR platform can be applied to native IgG1 antibodies, without antibody engineering, to yield highly homogeneous and stable ADCs via cysteine cross-linking. The resulting ADCs demonstrate a therapeutic index benefit compared to analogous conventional heterogeneous ADCs.
Citation Format: Jean-Francois Haeuw, Frederic Marion, Cyrille Dreyfus, Barbara Akla, Laurence Zanna, Marie-Claire Janin, Charlotte Beau-Larvor, Marion Millet, Martine Farrie, Alain Batton, Celine Thuilliez, Alain Beck, Sarah Cianferani, Thierry Clerc, Philippe Maillos, Nathalie Corvaia. Development of a new site-specific sulfomaleimide rebridging platform for the generation of homogeneous, stable, potent and safe antibody drug conjugates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4054.