Abstract 2967: In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs)
Authors: Harris Luke , Lanieri Leanne , Ponte Jose , Maloney Erin , Bartle Laura , Ab Olga , Costoplus Juliet , Rui Lingyun , Pinkas Jan , Chari Ravi , Keating Thomas , Tavares Daniel , Fishkin Nathan 
Published: July 2016

Abstract
Site-specific attachment of cell-killing agents to antibodies directed against tumor-associated antigens has continued to be an active area of innovation in the field of ADCs. Most reports focus on homogeneous ADCs that have a DAR (cytotoxic molecules per antibody ratio) of 2. Here we describe the preparation, biochemical characterization, and biological evaluation of ADCs made through conjugation of maytansinoids (DM1, DM4) to aldehydes derived from chemically oxidized N-terminal serines (SeriMab) engineered onto the antibody heavy chain (2 DAR) or both light and heavy chain simultaneously (4 DAR). ADCs prepared with a non-cleavable linker or a cleavable disulfide linker were homogeneous 2 or 4 DAR by MS analysis, and were produced in high yield with a monomer content of &gt;98%. Despite conjugation at the N-termini of both the light and heavy chain variable regions, FACS analysis showed the 4 DAR SeriMab conjugates maintained binding to the target antigen.
The ADCs showed antigen-specific potency in vitro on a panel of target-expressing cancer cell lines. In the disulfide cleavable linker series, the 2 DAR SeriMab conjugate was 2-5 fold less active than lysine-conjugated Ab-SPDB-DM4 (3.4 DAR), while the 4 DAR SeriMab conjugate was comparably active on an antibody basis. The SeriMab conjugates also displayed strong bystander killing. Surprisingly, in the non-cleavable linker series, the 2 DAR SeriMab conjugate was up to 17-fold more active (depending on cell line) than lysine-conjugated Ab-SMCC-DM1 (3.5 DAR), and the 4 DAR SeriMab conjugate was up to 100-fold more potent than the SMCC-DM1 conjugate on an antibody concentration basis. In a P-gp-positive multi-drug resistant cell line, the non-cleavable 4 DAR SeriMab-maytansinoid conjugate was highly active while the 2 DAR SeriMab ADCs and lysine-conjugated maytansinoid ADCs were &gt;100-fold less potent.
The unique oxime bond formed with the non-cleavable SeriMab-maytansinoid conjugate was found to be stable in circulation in mice for &gt;3 days as assayed by affinity capture LC-MS. Polar carboxylic acid containing metabolites were identified which may lead to high cellular retention of maytansinoid species in cancer cells, yielding higher in vitro potency than lysine-linked ADCs in some cell lines.
The in vivo anti-tumor activity of disulfide cleavable 2 and 4 DAR SeriMab-DM4 ADCs was evaluated in a clinically relevant cancer xenograft model. The 4 DAR conjugate was active at 60 μg/kg (maytansinoid payload dose) and was more active than the 2 DAR conjugate at this same payload dose.
Using the SeriMab conjugation platform we show that in vitro and in vivo activity of site-specific ADCs can be dependent on amount of cytotoxic agent attached per antibody.
Citation Format: Luke Harris, Leanne Lanieri, Jose Ponte, Erin Maloney, Laura Bartle, Olga Ab, Juliet Costoplus, Lingyun Rui, Jan Pinkas, Ravi Chari, Thomas Keating, Daniel Tavares, Nathan Fishkin. In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2967.