Abstract 644: Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates
Authors: Ma Dangshe , Jin Fang , Barletta Frank , Hu George , Tumey Nathan , Zhang Haige , He Tao , Sousa Eric , Charti Manoj , Khadke Kiran , Lucas Judy , Ferguson Darren , Brown Christoper , Ma Weijun , Gatto Scott , Brady William , Graziani Edmund , Gerber Hans-Peter , Sapra Puja , Tchistikova Lioudmila 
Published: August 2015

Abstract
Site-specific conjugation of toxic payload to antibody yields homogeneous ADCs resulting in improved therapeutic index over conventional conjugation. To understand the role of conjugation site on pharmacokinetics, off-target toxicity, cysteine(cys) was engineered into a humanized anti-IL13Rα2 antibody, hAB08, at various sites within the IgG1/k constant domains. Single cys mutants L443C and Q347C with DAR 2, and double cys mutants K392C+L443C, L443C/kK183C and Q347C/kK183C with DAR 4 were conjugated using auristatin 0101 via maleimidocapronic - valine-citruline-p-aminobenzyloxycarbonyl (vc, cleavable linker). All cys mutants and their conjugates maintained their binding properties compared to parental hAb08. Cysteine substitution sites were selected based on conjugation efficiency and in vitro stability. In vivo efficacy was evaluated in mouse xenograph tumor models. Pharmacokinetics (PK) and off target toxicity (TOX) were assessed in both mouse and rat in vivo studies. Our data demonstrate that that all site specific conjugates show improved in vitro stability, comparable or better in vivo efficacy in mice xenograph models and in vivo stability (ratio of ADC-AUC/Ab-AUC) compared to conventional conjugates. However PK and off-target toxicity of antibody drug conjugates had been significantly impacted by conjugation site and animal species. L443 mutants show higher ADC-AUC, lower clearance and longer half life than Q347 mutants in mice while a reverse TK behavior was observed in rat. All site specific conjugates show higher dose tolerance and less off-target toxicity relative to conventional conjugates in rat. This off-target toxicity improvement depends on sites of conjugation. The L443 mutants exhibited a better off-target TOX profile relative to Q347 mutants. Taken together, site-specific conjugation improved in vivo efficacy and off-target toxicity relative to conventionally conjugated ADC. PK differences and off-target toxicity improvements depend on conjugation sites and animal species.
Note: This abstract was not presented at the meeting.
Citation Format: Dangshe Ma, Fang Jin, Frank Barletta, George Hu, Nathan Tumey, Haige Zhang, Tao He, Eric Sousa, Manoj Charti, Kiran Khadke, Judy Lucas, Darren Ferguson, Christoper Brown, Weijun Ma, Scott Gatto, William Brady, Edmund Graziani, Hans-Peter Gerber, Puja Sapra, Lioudmila Tchistikova. Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates. [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 644. doi:10.1158/1538-7445.AM2015-644