Abstract 5691: Novel site-specific antibody drug conjugates based on novel amino acid incorporation technology have improved pharmaceutical properties over conventional antibody drug conjugates
Authors: Sapra Puja Sapra , Tchistiakova Lioudmila , Dushin Russell , Leal Mauricio , DiJoseph John , Veneziale Robert , Feyfant Eric , Gerber HansPeter , Sun Ying , Manibusan Anthony , Lu Yingchun , Sellers Aaron , Phuong Trung , DeGuzman Michael , Ensari Semsi , Barnett Barney , Cho Ho , Tian Feng 
Published: August 2012

We demonstrate here for the first time generation of ADCs by using EuCODETM technology that site-specifically incorporates novel amino acids into proteins in mammalian cells. Conventional conjugation strategies for making ADCs rely on conjugating the payload to the antibody via the side chains of lysines or cysteines: this procedure produces a heterogeneous mixture of ADCs exhibiting a range of biological activities. We believe that making ADCs using the novel amino acid incorporation technology (NDCs) will enable production of homogenous site specific conjugates that may have improved pharmaceutical properties, and simplified analytics compared to ADCs made using conventional conjugation strategies. 5T4 is an oncofetal antigen, that is selectively overexpressed on various cancers and is rapidly internalized upon antibody binding. Using anti-5T4 antibody (A1) as a model system, NDCs were made that contain the novel amino acid, para-acetylphenylalanine (pAF) at designated sites of the antibody molecule. Toxin payload, monomethyl auristatin D (MMAD), a potent tubulin inhibitor, was then conjugated via oxime linkages to the two engineered pAF residues in the anti-5T4 antibody to give homogenous NDCs with antibody:drug ratio of 1:2. Compared to the native form of the A1 antibody, pAF site-specific mutants and NDC conjugates made thereof do not alter the antibody binding and internalization kinetics on 5T4 positive tumor cells. Further, NDCs preserve the cytotoxicity observed with conventional ADCs. In vivo, NDCs demonstrated substantially improved pharmacokinetics in mice relative to conventional ADCs. For example, the mean systemic exposure for the NDC with a non-cleavable linker (nc) (A1-HS115pAF-ncMMAD) was significantly greater (2-4 fold) than that of A1-mcMMAD ADC (made using conventional cysteine-conjugation strategy). Most importantly, the NDCs had similar or better PK compared to the unconjugated Ab, suggesting that by using NDC technology, the payload is not lost from the Ab in circulation. In efficacy experiments, we compared NDCs and conventional ADCs head to head, using the same payload-linker. In two xenograft models, NDCs demonstrated equivalent or slightly better efficacy compared to conventional ADCs. Further, in a single dose rat toxicology studies, NDCs were well tolerated up to 90 mg/kg, the highest dose tested. To demonstrate the scalability of NDC production, a stable CHO clone for the corresponding mutated Ab of NDC HA114pAF was created and shown to support antibody production at &gt; 1 g/L using a fed-batch process. In summary, NDCs demonstrated improved in vivo efficacy and pharmacokinetic stability relative to ADCs prepared using conventional conjugation approaches. Based on these preclinical data, NDC technology could serve as another avenue for making site specific homogenous ADCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5691. doi:1538-7445.AM2012-5691