184 VOLUME 30 NUMBER 2 FEBRUARY 2012 NATURE BIOTECHNOLOGY LETTERS The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent- accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent- accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface. Antibody conjugates are generated by chemically linking a cysteine or lysine residue to potent chemotherapeutic drugs or other func- tional moieties by means of a variety of linkers 1–7 . Thiol-maleimide chemistry is a commonly used method, whereby a cysteine residue, either native or engineered, is used for conjugation 8–11 . Antibody conjugates for therapeutic applications must satisfy demanding criteria concerning specificity, safety and stability to achieve effec- tive delivery of the payload to the target. All three components— antibody, linker and payload—play critical roles in defining target specificity, the degree of stability and mechanism of action, respec- tively. Indeed, the in vivo stability and efficacy of antibody conjugates can be improved by optimizing the linker 9,12,13 and by selecting the appropriate antibody and payload 14,15 . However, additional contrib- uting factors, such as the influence of an antibody’s chemical and structural properties on the neighboring conjugation site, have not been thoroughly investigated. We chose antibody-drug conjugates (ADCs) and antibody- fluorophore conjugates (AFCs) as models to dissect the functional impact of the conjugation site on stability and biological activity. We have recently reported the generation of antibodies with cysteine residues engineered (referred to as THIOMABs) into the IgG heavy chain (HC-A114C) that provide reactive thiols for conjugation to the auristatin and maytansine classes of cytotoxic drugs 11,16 . These engineered ADCs displayed reduced liver and bone marrow toxicity compared to ADCs with the same drugs conjugated through either cysteine sulfhydryl groups activated by reducing interchain disulfide bonds or lysine side-chain amines 11,16 . Here we explored additional conjugation sites in the light chain (LC) 11 , heavy chain-Fab region (HC) 11 and heavy chain-Fc region (Fc) of trastuzumab (Herceptin) to generate THIOMAB variants for coupling to thiol-reactive linkers (Supplementary Fig. 1). Based on structural modeling, we selected three thio-trastuzumab variants (LC-V205C, HC-A114C and Fc-S396C) with conjugation sites differing in their local structural environments. Fc-S396C was predicted to have the highest fractional solvent accessibility, whereas both HC-A114C and LC-V205C variants were predicted to be in partially buried regions (Fig. 1a). The LC-V205C conjugation site appears to be located in a positively charged environment, whereas HC-A114C and Fc-S396C are in a relatively neutral environment (Fig. 1b). All three thio-trastuzumab variants showed cell-binding and internalization properties similar to that of native trastuzumab, indicating that the engineered cysteine residues do not alter the func- tional properties of the antibody (Supplementary Fig. 2). To assess their relative therapeutic activity, we conjugated mono- methyl auristatin E (MMAE) to the three thio-trastuzumab vari- ants by a maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (MC-vc-PAB) linker. The resulting ADCs are abbreviated as thio-trastuzumab-MC-vc-MMAE. Liquid chromatography– mass spectrometry (LC-MS) analysis confirmed that all three thio-trastuzumab-MC-vc-MMAE conjugates contained 1.7–1.9 drugs per antibody (Supplementary Table 1). Hydrophobic interac- tion chromatographic analysis revealed similar compositions of the Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates Ben-Quan Shen 1,2 , Keyang Xu 1,2 , Luna Liu 1 , Helga Raab 1 , Sunil Bhakta 1 , Margaret Kenrick 1 , Kathryn L Parsons-Reponte 1 , Janet Tien 1 , Shang-Fan Yu 1 , Elaine Mai 1 , Dongwei Li 1 , Jay Tibbitts 1 , Jakub Baudys 1 , Ola M Saad 1 , Suzie J Scales 1 , Paul J McDonald 1 , Philip E Hass 1 , Charles Eigenbrot 1 , Trung Nguyen 1 , Willy A Solis 1 , Reina N Fuji 1 , Kelly M Flagella 1 , Darshana Patel 1 , Susan D Spencer 1 , Leslie A Khawli 1 , Allen Ebens 1 , Wai Lee Wong 1 , Richard Vandlen 1 , Surinder Kaur 1 , Mark X Sliwkowski 1 , Richard H Scheller 1 , Paul Polakis 1 & Jagath R Junutula 1 1 Genentech Inc., 1 DNA Way, S. San Francisco, California, USA. 2 These authors contributed equally to this work. Correspondence should be addressed to J.R.J. (jagath@gene.com). Received 8 July 2011; accepted 20 December 2011; published online 22 January 2012; doi:10.1038/nbt.2108 npg © 2012 Nature America, Inc. All rights reserved.