As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build “design rules” that will enable more efficient prosecution of next-generation ADC discovery programs.
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Thomas A, Teicher BA, Hassan R. Antibody-drug conjugates for cancer therapy. Lancet Oncol. 2016;17(6):e254–62.
Panowski S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014;6(1):34–45.
Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Wong WL, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol. 2008;26:925–32.
Strop P, Liu S-H, Dorywalska M, Delaria K, Dushin RG, Tran T-T, Ho W-H, Farias S, Casas MG, Abdiche Y, Zhou D, Chandrasekaran R, Samain C, Loo C, Rossi A, Rickert M, Krimm S, Wong T, Chin SM, Yu J, Dilley J, Chaparro-Riggers J, Filzen GF, O’Donnell CJ, Wang F, Myers JS, Pons J, Shelton DL, Rajpal A. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol (Oxford, U K). 2013;20:161–7.
Shen B-Q, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, Parsons-Reponte KL, Tien J, Yu S-F, Mai E, Li D, Tibbitts J, Baudys J, Saad OM, Scales SJ, McDonald PJ, Hass PE, Eigenbrot C, Nguyen T, Solis WA, Fuji RN, Flagella KM, Patel D, Spencer SD, Khawli LA, Ebens A, Wong WL, Vandlen R, Kaur S, Sliwkowski MX, Scheller RH, Polakis P, Junutula JR. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol. 2012;30:184–9.
Tumey LN, Leverett CA, Vetelino B, Li F, Rago B, Han X, Loganzo F, Musto S, Bai G, Sukuru SC, Graziani EI, Puthenveetil S, Casavant J, Ratnayake A, Marquette K, Hudson S, Doppalapudi VR, Stock J, Tchistiakova L, Bessire AJ, Clark T, Lucas J, Hosselet C, O’Donnell CJ, Subramanyam C. Optimization of tubulysin antibody-drug conjugates: a case study in addressing ADC metabolism. ACS Med Chem Lett. 2016;7:977–82.
Xu K, Liu L, Saad OM, Baudys J, Williams L, Leipold D, Shen B, Raab H, Junutula JR, Kim A, Kaur S. Characterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography-mass spectrometry. Anal Biochem. 2011;412:56–66.
Axup Jun Y, Bajjuri Krishna M, Ritland M, Hutchins Benjamin M, Kim Chan H, Kazane Stephanie A, Halder R, Forsyth Jane S, Santidrian Antonio F, Stafin K, Lu Y, Tran H, Seller Aaron J, Biroc Sandra L, Szydlik A, Pinkstaff Jason K, Tian F, Sinha Subhash C, Felding-Habermann B, Smider Vaughn V, Schultz PG. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A. 2012;109:16101–6.
Rashidian M, Dozier JK, Distefano MD. Enzymatic labeling of proteins: techniques and approaches. Bioconjug Chem. 2013;24:1277–94.
Hamblett KJ, Senter PD, Chace DF, Sun MMC, Lenox J, Cerveny CG, Kissler KM, Bernhardt SX, Kopcha AK, Zabinski RF, Meyer DL, Francisco JA. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res. 2004;10:7063–70.
Adem YT, Schwarz KA, Duenas E, Patapoff TW, Galush WJ, Esue O. Auristatin antibody drug conjugate physical instability and the role of drug payload. Bioconjug Chem. 2014;25:656–64.
Zhao RY, Wilhelm SD, Audette C, Jones G, Leece BA, Lazar AC, Goldmacher VS, Singh R, Kovtun Y, Widdison WC, Lambert JM, Chari RVJ. Synthesis and evaluation of hydrophilic linkers for antibody-maytansinoid conjugates. J Med Chem. 2011;54:3606–23.
Stimmel JB, Merrill BM, Kuyper LF, Moxham CP, Hutchins JT, Fling ME, Kull Jr FC. Site-specific conjugation on serine right-arrow cysteine variant monoclonal antibodies. J Biol Chem. 2000;275:30445–50.
Lyons A, King DJ, Owens RJ, Yarranton GT, Millican A, Whittle NR, Adair JR. Site-specific attachment to recombinant antibodies via introduced surface cysteine residues. Protein Eng. 1990;3:703–8.
Puthenveetil S, Loganzo F, He H, Dirico K, Green M, Teske J, Musto S, Clark T, Rago B, Koehn F, Veneziale R, Falahaptisheh H, Han X, Barletta F, Lucas J, Subramanyam C, O’Donnell CJ, Tumey LN, Sapra P, Gerber HP, Ma D, Graziani EI. Natural product splicing inhibitors: a new class of antibody-drug conjugate (ADC) payloads. Bioconjug Chem. 2016;27:1880–8.
Maderna A, Doroski M, Subramanyam C, Porte A, Leverett CA, Vetelino BC, Chen Z, Risley H, Parris K, Pandit J, Varghese AH, Shanker S, Song C, Sukuru SCK, Farley KA, Wagenaar MM, Shapiro MJ, Musto S, Lam M-H, Loganzo F, O’Donnell CJ. Discovery of cytotoxic Dolastatin 10 analogues with N-terminal modifications. J Med Chem. 2014;57:10527–43.
Wei C, Zhang G, Clark T, Barletta F, Tumey LN, Rago B, Hansel S, Han X. Where did the linker-payload go? A quantitative investigation on the destination of the released linker-payload from an antibody-drug conjugate with a maleimide linker in plasma. Anal Chem (Washington, DC, U S). 2016;88:4979–86.
Krivov GG, Shapovalov MV, Dunbrack Jr RL. Improved prediction of protein side-chain conformations with SCWRL4. Proteins. 2009;77:778–95.
Ye JD, Tereshko V, Frederiksen JK, Koide A, Fellouse FA, Sidhu SS, Koide S, Kossiakoff AA, Piccirilli JA. Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc Natl Acad Sci U S A. 2008;105:82–7.
Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney Jr DW, Leahy DJ. Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature. 2003;421:756–60.
Spassov VZ. Yan LA fast and accurate computational approach to protein ionization. Protein Sci. 2008;17:1955–70.
Rago B, Clark T, King L, Zhang J, Tumey LN, Li F, Barletta F, Wei C, Leal M, Hansel S, Han X. Calculated conjugated payload from immunoassay and LC-MS intact protein analysis measurements of antibody-drug conjugate. Bioanalysis. 2016;8:2205–17.
Schlothauer T, Rueger P, Stracke JO, Hertenberger H, Fingas F, Kling L, Emrich T, Drabner G, Seeber S, Auer J, Koch S, Papadimitriou A. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies. MAbs. 2013;5:576–86.
Tessier PM, Jinkoji J, Cheng YC, Prentice JL, Lenhoff AM. Self-interaction nanoparticle spectroscopy: a nanoparticle-based protein interaction assay. J Am Chem Soc. 2008;130:3106–12.
Voynov V, Chennamsetty N, Kayser V, Wallny HJ, Helk B, Trout BL. Design and application of antibody cysteine variants. Bioconjug Chem. 2010;21:385–92.
Duppatla V, Gjorgjevikj M, Schmitz W, Kottmair M, Mueller TD, Sebald W. Enzymatic deglutathionylation to generate interleukin-4 cysteine muteins with free thiol. Bioconjug Chem. 2012;23:1396–405.
Dubowchik GM, Firestone RA, Padilla L, Willner D, Hofstead SJ, Mosure K, Knipe JO, Lasch SJ, Trail PA. Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem. 2002;13:855–69.
Tumey LN, Charati M, He T, Sousa E, Ma D, Han X, Clark T, Casavant J, Loganzo F, Barletta F, Lucas J, Graziani EI. Mild method for succinimide hydrolysis on ADCs: impact on ADC potency, stability, exposure, and efficacy. Bioconjug Chem. 2014;25:1871–80.
Lyon RP, Bovee TD, Doronina SO, Burke PJ, Hunter JH, Neff-LaFord HD, Jonas M, Anderson ME, Setter JR, Senter PD. Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nat Biotechnol. 2015;33:733–5.
Marshall DJ, Harried SS, Murphy JL, Hall CA, Shekhani MS, Pain C, Lyons CA, Chillemi A, Malavasi F, Pearce HL, Thorson JS, Prudent JR. Extracellular antibody drug conjugates exploiting the proximity of two proteins. Mol Ther. 2016;24:1760–70.
Lyon RP, Setter JR, Bovee TD, Doronina SO, Hunter JH, Anderson ME, Balasubramanian CL, Duniho SM, Leiske CI, Li F, Senter PD. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nat Biotechnol. 2014;32:1059–62.
We would like to gratefully acknowledge the work of Russell Dushin, Dahui Zhou, Matthew Doroski, Alex Porte, Hud Risley, Zecheng Chen, and Andreas Maderna for the preparation and identification of mcValCitPABC_06380101. Dosing of animals was performed by Judy Lucas and Christine Hosselet.
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Tumey, L.N., Li, F., Rago, B. et al. Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates. AAPS J 19, 1123–1135 (2017). https://doi.org/10.1208/s12248-017-0083-7
- antibody drug conjugate
- linker stability
- PK exposure
- plasma stability