Abstract 650: A high-throughput conjugation strategy for the selection of THIOMAB™ antibodies with desired properties for antibody-drug conjugation

Abstract THIOMAB™ antibody technology utilizes antibody engineering to replace endogenous amino acids of an antibody with cysteine to allow for site-selective conjugation. The technology enables the exploration of different drug attachment sites on the antibody thereby offering the potential to select antibody-drug conjugates (ADCs) with unique properties. Having a large number of antibody sites from which to choose may be desired for some applications. Many antibody sites are not suitable due to limited solvent accessibility or because drug attachment alters the native antibody properties. To identify those antibody sites most amenable to drug conjugation, we prepared 648 THIOMAB™ antibody variants of the anti-HER 2 antibody 4D5 by sequentially mutating every amino acid in the light and heavy chains to cysteine (excluding the endogenous cysteines). Each THIOMAB™ antibody variant was then conjugated to maleimido caproyl valine citrulline PABA MMAE and pyridyl disulfide MMAE in separate reactions. A high throughput on-bead Protein A conjugation and purification method was developed using a 96 well plate format. Complete reduction of the engineered cysteines and reoxidation of the endogenous cysteines, and drug to antibody ratios (DAR) were all determined by LC/MS. The concentration and aggregation status of each ADC were determined using automated liquid chromatography methods. Nearly 40% of the THIOMAB™ antibodies were found to be suitable for conjugation with DAR >1 and low aggregation levels. The other 60% failed due to a variety of factors including low drug incorporation, high aggregation levels, and incomplete re-oxidation of the endogenous cysteines. We found similar results with the maleimide and pyridyl disulfide derivatives suggesting our findings may be applicable to other auristatin derivatives with different sulfhydryl reactive chemistries. In summary, we have developed a high throughput conjugation strategy for THIOMAB™ antibodies and used it to identify the antibody sites most suitable for conjugation to auristatin derivatives. Citation Format: Rachana V. Ohri, Sunil Bhakta, Helga Raab, Richard Vandlen, Jagath Junutula, Hans Erickson. A high-throughput conjugation strategy for the selection of THIOMAB™ antibodies with desired properties for antibody-drug conjugation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 650. doi:10.1158/1538-7445.AM2015-650