Abstract 653: Antibody-drug conjugates: engineered N-terminal serine residues as a novel approach for site-specific conjugation
Authors: Tavares Daniel , Rui Lingyun , Ab Olga , Harris Luke , Maloney Erin , Keating Thomas , Chittenden Thomas , Fishkin Nathan 
Published: August 2015

Emerging data from the growing number of antibody-drug conjugates (ADCs) in clinical studies reflects the critical contributions from each component of the ADC. The tumor-targeting antibody and the cytotoxic effector molecule are recognized as key elements of an ADC, but increasingly the linkers and linkage mechanisms are being seen as critical for optimizing the design of an ADC based on the specific properties of the target antigen, tumor types, and clinical indications. Traditionally, antibodies have been conjugated to their cytotoxic payloads either at their lysine residues or through cysteine residues accessed through reduction of intrachain disulfide bonds. Recently a number of site-specific conjugation methods have been reported. Here we describe the construction of antibodies and antibody fragments with engineered N-terminal serine residues to facilitate a novel approach for site-specific antibody conjugation.

Serine residues can be engineered into at least four different N-terminal positions on an antibody. Serines are found at the N-termini of some naturally occurring human antibodies, but in most cases they must be introduced by either replacing the native N-terminal residue or by including an additional serine residue at the N-terminus. These modifications can be made on the light chain, heavy chain, or both to provide as many as 4 potential linkage sites per antibody.

Modification at the N-terminus situates the linked cytotoxic molecule well outside of the antibody's target binding sites in the variable region complementarity determining regions (CDRs). Molecular modeling demonstrates that this is the case whether the linkage is on either the light or heavy chain N-termini. ELISA and flow cytometry binding data demonstrated that changing the N-terminal residue to serine itself had no impact on the binding properties of the parent antibody. Likewise, ADCs utilizing the N-terminal serine for conjugation had similar binding properties compared with lysine or engineered cysteine conjugates of the same antibody. Finally, in vitro cytotoxicity assays demonstrated that N-terminal serine conjugates were at least as potent as conventional ADCs against antigen-expressing tumor cells.

The utilization of a variable region framework residue means that the same N-terminal serines used for antibody conjugation can be applied to antibody fragments. Due to the size and residue usage limitations of antibody fragments, conjugation of hydrophobic, cytotoxic agents to these molecules can be challenging. We have built and tested N-terminal serine conjugates with Fabs, scFvs, and sdAbs, establishing the utility of this approach across a wide range of antibody fragment formats.

In conclusion, conjugation of engineered antibody N-terminal serine residues represents a promising approach to site-specific ADCs that is worthy of further development.

Citation Format: Daniel Tavares, Lingyun Rui, Olga Ab, Luke Harris, Erin Maloney, Thomas Keating, Thomas Chittenden, Nathan Fishkin. Antibody-drug conjugates: engineered N-terminal serine residues as a novel approach for site-specific 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 653. doi:10.1158/1538-7445.AM2015-653