To overcome stability and heterogeneity issues of antibody–drug conjugates (ADCs) produced with existing bioconjugation technologies incorporating a maleimide motif, we developed McSAF Inside, a new technology based on a trifunctionalized di­(bromomethyl)­pyridine scaffold. Our solution allows the conjugation of a linker-payload to previously reduced interchain cysteines of a native antibody, resulting in disulfide rebridging. This leads to highly stable and homogeneous ADCs with control over the drug-to-antibody ratio (DAR) and the linker-payload position. Using our technology, we synthesized an ADC, MF-BTX-MMAE, built from anti-CD30 antibody cAC10 (brentuximab), and compared it to Adcetris, the first line treatment against CD30-positive lymphoma, in a CD30-positive lymphoma model. MF-BTX-MMAE displayed improved DAR homogeneity, with a solid batch-to-batch reproducibility, as well as enhanced stability in thermal stress conditions or in the presence of a free thiol-containing protein, such as human serum albumin (HSA). MF-BTX-MMAE showed antigen-binding, in vitro cytotoxicity, in vivo efficacy, and tolerability similar to Adcetris. Therefore, in accordance with current regulatory expectations for the development of new ADCs, McSAF Inside technology gives access to relevant ADCs with improved characteristics and stability.

Antibody−drug conjugates (ADCs) are the spearhead of targeted therapies. According to the technology used, the conjugation of a cytotoxic drug to an antibody can produce suboptimal heterogeneous species, impacting the overall efficacy. Herein, we describe the synthesis of HER2targeting ADCs with three disulfide rebridging heads, allowing homogeneous and site-specific bioconjugation: dibromomaleimide (DBM), dithiomaleimide (DTM), and hybrid thiobromomaleimide (TBM) chemical bricks to combine the properties of both previously used heads. The primary purpose of this study was to compare the reactivity of these three chemical bricks in the bioconjugation process. Then, the resulting ADCs were evaluated in terms of physicochemical stability, binding, and biological activity. We have demonstrated that the higher percentage of a drug-to-antibody ratio of 4 was obtained with TBM. Additionally, the reaction time was drastically reduced with TBM in comparison to DTM. The three ADCs showed good binding to HER2 and in vitro cytotoxicity, which validate the TBM structure as an attractive alternative scaffold for rebridging bioconjugation.

With three clinically approved antibody-drug conjugates targeting HER2, this target is clearly identified to be of interest in oncology. Moreover, the advent of new bioconjugation technologies producing site-specific homogenous conjugates led to the opportunity of developing new medicines linking antibodies and payloads. Here, a new relevant HER2-targeting ADC was obtained by the conjugation of monomethyl auristatin E onto trastuzumab using McSAF Inside bioconjugation technology. The antibody-drug conjugate formed presented an average drug-to-antibody ratio of 4 with a high homogeneity and an excellent stability especially when incubated with human serum albumin or in human plasma. Moreover, it demonstrated a strong efficacy in an HER2 xenograft tumor model in mice, superior to the clinically approved antibody-drug conjugate ado-trastuzumab emtansine, with a complete tumor regression observed both macroscopically and microscopically demonstrating its therapeutic potential.

Introduction: The Antibody-Drug Conjugates (ADCs)-based therapy has revealed a field of perspectives by increasing the specificity and the efficacy of the anti-tumoral drug treatment. McSAF is a company focused in bio-organic and bioconjugates chemistry and has developed a new linker technology solution, allowing to improve ADCs homogeneity and stability resulting in a higher anti-tumoral effect on a HER2-positive breast tumor model (BT-474 cell line) and a CD30-positive non-Hodgkin's lymphoma model (Karpas-299 cell line). Methods: The anti-tumoral activity of McSAF-ADCs was tested both in vitro and in vivo. In both cell line models, an MTS-based cell viability assay has been performed in presence of the standard of care (SOC, Kadcyla® (T-DM1) = Trastuzumab emtansine, or Adcetris® = Brentuximab vedotin) or the equivalent McSAF-ADCs at different concentrations for 72 to 96 hours. In vivo studies were conducted with xenograft models of HER2-positive tumors in Balb-c nude mice and CD30-positive lymphoma in CB17 SCID mice. After tumor induction, animals bearing well-established tumors (≈100-200 mm3) were randomized and treated once or twice with vehicle, SOC or McSAF-ADCs at one or two doses. During the study course, the animals were monitored for several weeks for their behavior, body weight and tumor volume. Results: In the HER2-positive breast tumor model, in vitro experiments revealed a higher cytotoxicity of MF-TTZ-MMAE (IC50 = 1 nM) on BT-474 cell line compared to the SOC (IC50 ≈ 100 nM). In vivo, the treatment with MF-TTZ-MMAE was well tolerated at all tested doses (1 and 5 mg/kg). At 5 mg/kg, the MF-TTZ-MMAE compound induced a full regression of tumors after second treatment. At the end of the study, all animals treated with MF-TTZ-MMAE were still tumor free, seven weeks after end of treatment, which was not observed on animals treated with T-DM1 (only 25% tumor-free animals). In the CD30-positive non-Hodgkin's lymphoma model, the MTS assay revealed similar efficacy for brentuximab vedotin and MF-BTX-MMAE (IC50 ≈ 0.1 nM) on Karpas-299 cell line. In vivo, the treatment with MF-BTX-MMAE was well tolerated at all tested doses (0.5 and 1 mg/kg). The single treatment with MF-BTX-MMAE had a marked, dose dependent, anti-tumoral efficacy with 40% and 100% of animals showing complete regression at the end of the study (9 weeks after treatment) for doses of 0.5 mg/kg and 1 mg/kg respectively. And again, the treatment with MF-BTX-MMAE at 0.5 mg/kg resulted in a higher tumor growth inhibition and better survival than with the clinical equivalent ADC Adcetris® at the equivalent dose. Conclusion: Altogether, these promising results reveal that improvement of ADCs stability and homogeneity leads to a higher anti-tumoral activity as tested both in vitro and in vivo HER2-positive and CD30-positive tumor models. Citation Format: Jean-Francois Mirjolet, Audrey Bertaux, Samira Benhamouche-Trouillet, Pascal Grondin, Ambrine Sahal, Guillaume Serin, Ludovic Juen, Christine Baltus, Camille Gély, Ofelia Feuillâtre, Audrey Desgranges, Marie-Claude Viaud-Massuard, Camille Martin. ADCs optimization lead to a significant anti-tumoral activity in lymphoma and breast tumor xenograft mouse models [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 4115.