Dimasi (2019) Design and characterization of homogenous antibody-drug conjugates with a drug-to-antibody ratio of one prepared using an engineered antibody and a dual-maleimide pyrrolobenzodiazepine dimer, mAbs, 11:3, 500-515, ABSTRACTMost strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent antitumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two. ARTICLE HISTORY

We describe the characterization of antigen binding fragments (Fab)–drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross‐linkers, are a class of payloads used in antibody–drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug‐to‐Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti‐HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti‐HER2 trastuzumab Fab‐rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen‐dependent cancer cell‐killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab–pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.

Bispecific antibody (bsAb) applications have exponentially expanded with the advent of molecular engineering strategies that have addressed many of the initial challenges, including improper light chain pairing, heterodimer purity, aggregation, and pharmacokinetics. However, the lack of high-throughput methods for the generation of monovalent bsAbs has resulted in a bottleneck that has hampered their therapeutic evaluation, as current technologies can be cost-prohibitive and impractical. To address this issue, we incorporated single-matched point mutations in the CH3 domain to recapitulate the physiological process of human IgG4 Fab-arm exchange to generate monovalent bsAbs. Furthermore, we utilized the substitutions H435R and Y436F in the CH3 domain of IgG1, which incorporates residues from human IgG3, thus ablating protein A binding. By exploiting this combination of mutations and optimizing the reduction and reoxidation conditions for Fab arm exchange, highly pure monovalent bsAbs can be rapidly purified directly from combined culture media using standard protein A purification. This methodology, reported herein for the first time, allows for the high-throughput generation of monovalent bsAbs, thus increasing the capacity for evaluating monovalent bsAb iterations for therapeutic potential.

Antibody drug conjugates (ADCs) combine a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer. ADCs bearing non-cleavable drug-linkers such as the pyrrolobenzodiazepine (PBD) SG3376 or the maytansinoid DM1 rely upon lysosomal degradation of the antibody for release of the cytotoxic warheads, which must then escape the lysosome and bind to their intracellular targets. We sought to identify the mechanism of lysosomal transport of these membrane-insoluble warheads and have recently reported that the lysosomal transporter SLC46A3 is required for cytotoxic activity of ADCs bearing SG3376 and DM1, and that loss of SLC46A3 is a mechanism of acquired resistance to Trastuzumab-DM1 (T-DM1). Here we explore the potential translational relevance of SLC46A3. Analysis of SLC46A3 mRNA expression across several tumors showed that breast cancer cell lines and primary tumor samples have relatively high levels of SLC46A3 compared to samples derived from multiple myeloma (MM), esophageal, and upper aerodigestive tract malignancies, all of which are being targeted in clinical trials with non-cleavable DM1 ADCs. Deeper analysis indicates that levels of SLC46A3 are decreased in MM patients compared to healthy donors, and levels are further decreased in the relapsed/refractory setting compared to samples taken at diagnosis. We measured the expression of SLC46A3 in a panel of MM cell lines by RT-PCR and found that 5 of 9 (56%) had undetectable levels of SLC46A3. In vitro cytotoxicity assays demonstrate that SLC46A3-negative cells are resistant to B-cell maturation antigen (BCMA)-targeted ADCs prepared with non-cleavable DM1 and SG3376 warheads, but remain sensitive to the cleavable payload PBD SG3249 (Tesirine). Taken together, our results suggest that SLC46A3 may be a predictive response marker for ADCs bearing the non-cleavable warheads SG3376 and DM1, and this marker may be particularly useful in trials involving MM. Citation Format: Krista Kinneer, Yu-Tzu Tai, Sriram Sridhar, John Meekin, Sandrina Phipps, Ben Ruddle, Ryan Fleming, Nazzareno Dimasi, Ronald Herbst, Arnaud Tiberghien, Luke Masterson, Philip Howard, Kenneth Anderson, David Tice. The lysosomal transporter SLC46A3 is a potential predictive biomarker for antibody-drug conjugates bearing the non-cleavable warheads SG3376 and DM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 567.