Abstract
Antibody drug conjugates (ADCs) have begun to have a tremendous impact on the treatment of cancer and other pathological conditions. A current limitation in ADC development is that much effort and time is needed to fully optimise the combination of antibody, linker and drug. New linker strategies are required to ensure that more homogeneous and stable ADCs can be produced with more predictable in vivo behaviour without the need for extensive re-optimisation, especially if one component of the ADC is changed. In order to improve both the homogeneity and the stability of ADCs, we have developed linkers that allow site-specific drug conjugation based on bis-sulphones that covalently re-bridge reduced disulphide bonds. The bis-sulphone reagents comprise a drug, a linker and a bis-reactive conjugating moiety that is capable of undergoing reaction with both sulphur atoms derived from a reduced disulphide bond in antibodies and antibody fragments. We have demonstrated that the bis-sulphone-derived conjugates retain antigen-binding, are stable in serum and exhibit potent and antigen-selective cell killing in both in vitro and in vivo cancer models. Disulphide re-bridging conjugation is a general approach to prepare stable ADCs, which does not require the antibody to be recombinantly re-engineered for site-specific conjugation. The bis-sulphone linker-platform is being developed by Abzena plc under the trade name ThioBridgeâ„¢.
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References
Agarwal P, Bertozzi CR (2015) Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjug Chem 26(2):176–192
Alley SC, Benjamin DR, Jeffrey SC, Okeley NM, Meyer DL, Sanderson RJ, Senter PD (2008) Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug Chem 19(3):759–765
Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, Halder R, Forsyth JS, Santidrian AF, Stafin K, Lu Y, Tran H, Seller AJ, Biroc SL, Szydlik A, Pinkstaff JK, Tian F, Sinha SC, Felding-Habermann B, Smider VV, Schultz PG (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A 109(40):16101–16106
Badescu G, Bryant P, Bird M, Henseleit K, Swierkosz J, Parekh V, Tommasi R, Pawlisz E, Jurlewicz K, Farys M, Camper N, Sheng X, Fisher M, Grygorash R, Kyle A, Abhilash A, Frigerio M, Edwards J, Godwin A (2014) Bridging disulfides for stable and defined antibody drug conjugates. Bioconjug Chem 25(6):1124–1136
Balan S, Choi J, Godwin A, Teo I, Laborde CM, Heidelberger S, Zloh M, Shaunak S, Brocchini S (2007) Site-specific PEGylation of protein disulfide bonds using a three carbon bridge. Bioconjug Chem 18(1):61–76
Beck A (2014) Review of antibody-drug conjugates, methods in molecular biology series: a book edited by Laurent Ducry. MAbs 6(1):30–33
Behrens CR, Liu B (2014) Methods for site-specific drug conjugation to antibodies. MAbs 6(1):46–53
Betz SF (1993) Disulfide bonds and the stability of globular proteins. Protein Sci 2:1551–1558
Brocchini S, Balan S, Godwin A, Choi J, Zloh M, Shaunak S (2006) PEGylation of native disulfide bonds in proteins. Nat Protoc 1(5):2241–2252
Brocchini S, Godwin A, Balan S, Choi J, Zloh M, Shaunak S (2008) Disulfide bridged based PEGylation of proteins. Adv Drug Deliv Rev 60(1):3–12
Bryant P, Pabst M, Badescu G, Bird M, McDowell W, Jamieson E, Swierkosz J, Jurlewicz K, Tommasi R, Henseleit K, Sheng X, Camper N, Manin A, Kozakowska K, Peciak K, Laurine E, Grygorash R, Kyle A, Morris D, Parekh V, Abhilash A, Choi JW, Edwards J, Frigerio M, Baker MP, Godwin A (2015) In vitro and in vivo evaluation of cysteine rebridged trastuzumab-MMAE antibody drug conjugates with defined drug-to-antibody ratios. Mol Pharm 12(6):1872–1879
Bryden F, Maruani A, Savoie H, Chudasama V, Smith ME, Caddick S, Boyle RW (2014) Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment. Bioconjug Chem 25(3):611–617
Castaneda L, Maruani A, Schumacher FF, Miranda E, Chudasama V, Chester KA, Baker JR, Smith ME, Caddick S (2013) Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation. Chem Commun (Camb) 49(74):8187–8189
Chari RV, Miller ML, Widdison WC (2014) Antibody-drug conjugates: an emerging concept in cancer therapy. Angew Chem 53(15):3796–3827
Choi J, Godwin A, Balan S, Bryant P, Cong Y, Pawlisz E, Porssa M, Rumpf N, Singh R, Powell K, Brocchini S (2009) Rebridging disulphides: site-specific PEGylation by sequential bis-alkylation. In: Veronese FM (ed) Protein PEGylation, basic science and biological applications. Springer, Basel, pp 47–74
Chudasama V, Smith ME, Schumacher FF, Papaioannou D, Waksman G, Baker JR, Caddick S (2011) Bromopyridazinedione-mediated protein and peptide bioconjugation. Chem Commun (Camb) 47(31):8781–8783
Cline DJ, Redding SE, Brohawn SG, Psathas JN, Schneider JP, Thorpe C (2004) New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability. Biochemistry 43(48):15195–15203
Darby N, Creighton TE (1995) Disulfide bonds in protein folding and stability. Methods Mol Biol 40:219–252
Darby N, Creighton TE (1997) Probing protein folding and stability using disulfide bonds. Mol Biotechnol 7(1):57–77
Debaene F, Boeuf A, Wagner-Rousset E, Colas O, Ayoub D, Corvaia N, Van Dorsselaer A, Beck A, Cianferani S (2014) Innovative native MS methodologies for antibody drug conjugate characterization: high resolution native MS and IM-MS for average DAR and DAR distribution assessment. Anal Chem 86(21):10674–10683
del Rosario RB, Wahl RL, Brocchini SJ, Lawton RG, Smith RH (1990) Sulfhydryl site-specific cross-linking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation cross-link reagent. Bioconjug Chem 1(1):51–59
Dennler P, Chiotellis A, Fischer E, Bregeon D, Belmant C, Gauthier L, Lhospice F, Romagne F, Schibli R (2014) Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjug Chem 25(3):569–578
Dokter W, Ubnik R, van der Lee M, van der Vleuten M, van Achterberg T, Jacobs D, Loosveld E, van der Dobbelsteen D, Egging D, Mattaar E, Groothius P, Beusker P, Coumans R, Elgersma R, Menge W, Joosten J, Spijker H, Huijbregts T, de Groot V, Eppink M, de Roo G, Verheijden G, Timmers M (2014) Preclinical profile of the HER2-targeting ADC SYD983/SYD985: introduction of a new duocarmycin-based linker-drug platform. Mol Cancer Ther 13(11):2618–2629
Doronina SO, Mendelsohn BA, Bovee TD, Cerveny CG, Alley SC, Meyer DL, Oflazoglu E, Toki BE, Sanderson RJ, Zabinski RF, Wahl AF, Senter PD (2006) Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem 17(1):114–124
Fass D (2012) Disulfide bonding in protein biophysics. Annu Rev Biophys 41:63–79
Flemming A (2014) Antibody engineering: fine-tuning antibody-drug conjugates. Nat Rev Drug Discov 13(3):178
Francisco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF, Rejniak SX, Gordon KA, DeBlanc R, Toki BE, Law CL, Doronina SO, Siegall CB, Senter PD, Wahl AF (2003) cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood 102(4):1458–1465
Gorovits B, Alley SC, Bilic S, Booth B, Kaur S, Oldfield P, Purushothama S, Rao C, Shord S, Siguenza P (2013) Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists antibody-drug conjugate working group position paper. Bioanalysis 5(9):997–1006
Hallam TJ, Smider VV (2014) Unnatural amino acids in novel antibody conjugates. Future Med Chem 6(11):1309–1324
Hallam TJ, Wold E, Wahl A, Smider VV (2015) Antibody conjugates with unnatural amino acids. Mol Pharm 12(6):1848–1862
Hamblett KJ, Mayer DL, Chace DF, Zabinsky RF, Cerveny CG, Sun MM, Senter PD, Francisco JA (2004a) Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate. Proc Am Assoc Cancer Res 45:624
Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, Kissler KM, Bernhardt SX, Kopcha AK, Zabinski RF, Meyer DL, Francisco JA (2004b) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10(20):7063–7070
Han JC, Han GY (1994) A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal Biochem 220(1):5–10
Hogg PJ (2003) Disulfide bonds as switches for protein function. Trends Biochem Sci 28(4):210–214
http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Public_assessment_report/human/002455/WC500135054.pdf (2012) EMA Assessment report: Adcetris
Jeger S, Zimmermann K, Blanc A, Grünberg J, Honer M, Hunziker P, Struthers H, Schibli R (2010) Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew Chem Int Ed 49:9995–9997
Jones MW, Strickland RA, Schumacher FF, Caddick S, Baker JR, Gibson MI, Haddleton DM (2012) Polymeric dibromomaleimides as extremely efficient disulfide bridging bioconjugation and pegylation agents. J Am Chem Soc 134(3):1847–1852
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 (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26:925–932
Khalili H, Godwin A, Choi J, Lever R, Khaw PT, Brocchini S (2012) Comparative binding of disulfide-bridged PEG-Fabs. Bioconjug Chem 23(11):2262–2277
Khalili H, Godwin A, Choi J, Lever R, Brocchini S (2013) Fab-PEG-fab as a potential antibody mimetic. Bioconjug Chem 24(11):1870–1882
Liberatore FA, Comeau RD, McKearin JM, Pearson DA, Belonga BQ 3rd, Brocchini SJ, Kath J, Phillips T, Oswell K, Lawton RG (1990) Site-directed chemical modification and cross-linking of a monoclonal antibody using equilibrium transfer alkylating cross-link reagents. Bioconjug Chem 1(1):36–50
Liu H, May, K. (2012) DIsulfide bond structures of IgG molecules, structural variantions, chemical modifications and possible impacts to stability and biological function. MAbs 4(1):17–23
Liu H, Chumsae C, Gaza-Bulseco G, Hurkmans K, Radziejewski CH (2010) Ranking the susceptibility of disulfide bonds in human IgG1 antibodies by reduction, differential alkylation, and LC-MS analysis. Anal Chem 82(12):5219–5226
Mack F, Ritchie M, Sapra P (2014) The next generation of antibody drug conjugates. Semin Oncol 41(5):637–652
Marcoux J, Champion T, Colas O, Wagner-Rousset E, Corvaia N, Van Dorsselaer A, Beck A, Cianferani S (2015) Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate. Protein Sci 24(8):1210–1223
Marculescu C, Kossen H, Morgan RE, Mayer P, Fletcher SA, Tolner B, Chester KA, Jones LH, Baker JR (2014) Aryloxymaleimides for cysteine modification, disulfide bridging and the dual functionalization of disulfide bonds. Chem Commun (Camb) 50(54):7139–7142
Maruani A, Smith MEB, Miranda E, Chester KA, Chudasama V, Caddick S (2015) A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nat Commun 6:6645
Miao Z, Hong Y, Zhu T, Chucholowski AW (2013) Drug-conjugates, conjugation methods, and uses thereof. Google Patents
Narayan M (2012) Disulfide bonds: protein folding and subcellular protein trafficking. FEBS J 279(13):2272–2282
Nelson AL (2012) Antibody fragments: hope and hype. MAbs 2(1):77–83
Panowski S, Bhakta S, Raab H, Polakis P, Junutula JR (2014) Site-specific antibody drug conjugates for cancer therapy. MAbs 6(1):34–45
Powers G, Hudson PJ, Wheatcroft MP 2012 Design and production of multimeric antibody fragments, focused on diabodieswith enhanced clinical efficacy. Antibody Engineering. Chapter 39
Rohrer T (2012) Consideration for the safe and effective manufacturing of antibody drug conjugates. Chimica Oggi-Chemistry Today 30(5):76–79
Ryan CP, Smith ME, Schumacher FF, Grohmann D, Papaioannou D, Waksman G, Werner F, Baker JR, Caddick S (2011) Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis. Chem Commun (Camb) 47(19):5452–5454
Schumacher FF, Nobles M, Ryan CP, Smith ME, Tinker A, Caddick S, Baker JR (2011) In situ maleimide bridging of disulfides and a new approach to protein PEGylation. Bioconjug Chem 22(2):132–136
Schumacher F, Sanchania VA, Tolner B, Wright ZVF, Ryan CP, Smith MEB, Ward JM, Caddick S, Kay CWM, Aeppli G, Chester KA, Baker JR (2013) Homogeneous antibody fragment conjugation by disulfide bridging introduces ‘spinostics’. Sci Rep 3:1525–1532
Schumacher FF, Nunes JPM, Maruani A, Chudasama V, Smith MEB, Chester KA, Baker JR, Caddick S (2014) Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging. Org Biomol Chem 12:7261–7269
Senter PD (2009) Potent antibody drug conjugates for cancer therapy. Curr Opin Chem Biol 13(3):235–244
Shaunak S, Godwin A, Choi J, Balan S, Pedone E, Vijayarangam D, Heidelberger S, Teo I, Zloh M, Brocchini S (2006) Site-specific PEGylation of native disulfide bonds in therapeutic proteins. Nat Chem Biol 2(6):312–313
Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, Parsons-Reponte KL, Tien J, Yu SF, 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 (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol 30(2):184–189
Sievers EL, Senter PD (2013) Antibody-drug conjugates in cancer therapy. Annu Rev Med 64:15–29
Smith MEB, Schumacher FF, Ryan CP, Tedaldi LM, Papaioannou D, Waksman G, Caddick S, Baker JR (2010) Protein modification, bioconjugation, and disulfide bridging using bromomaleimides. J Am Chem Soc 132(6):1960–1965
Sochaj AM, Swiderska KW, Otlewski J (2015) Current methods for the synthesis of homogeneous antibody-drug conjugates. Biotechnol Adv. doi:10.1016/j.biotechadv.2015.05.001
Strop P, Liu SH, Dorywalska M, Delaria K, Dushin RG, Tran TT, Ho WH, 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 (2013) Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol 20(2):161–167
Strop P, Delaria K, Foletti D, Witt JM, Hasa-Moreno A, Poulsen K, Casas MG, Dorywalska M, Farias S, Pios A, Lui V, Dushin R, Zhou D, Navaratnam T, Tran TT, Sutton J, Lindquist KC, Han B, Liu SH, Shelton DL, Pons J, Rajpal A (2015) Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading. Nat Biotechnol 33(7):694–696
Sun MM, Beam KS, Cerveny CG, Hamblett KJ, Blackmore RS, Torgov MY, Handley FG, Ihle NC, Senter PD, Alley SC (2005) Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug Chem 16(5):1282–1290
Tian F, Lu Y, Manibusan A, Sellers A, Tran H, Sun Y, Phuong T, Barnett R, Hehli B, Song F, DeGuzman MJ, Ensari S, Pinkstaff JK, Sullivan LM, Biroc SL, Cho H, Schultz PG, DiJoseph J, Dougher M, Ma D, Dushin R, Leal M, Tchistiakova L, Feyfant E, Gerber HP, Sapra P (2014) A general approach to site-specific antibody drug conjugates. Proc Natl Acad Sci U S A 111(5):1766–1771
Trail PA (2013) Antibody drug conjugates as cancer therapeutics. Antibodies 2:113–129
Trivedi MV, Laurence JS, Siahaan TJ (2009) The role of thiols and disulfides on protein stability. Curr Protein Pept Sci 10(6):614–625
Vidarsson G, Dekkers G, Rispens T (2014) IgG subclasses and allotypes: from structure to effector functions. Front Immunol 5:520
Wahl AF, Donaldson KL, Mixan BJ, Trail PA, Siegall CB (2001) Selective tumor sensitization to taxanes with the mAb-drug conjugate cBR96-doxorubicin. Int J Cancer 93(4):590–600
Wakankar A, Chen Y, Gokarn Y, Jacobson FS (2011) Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs 3(2):161–172
Wedemeyer WJ, Welker E, Narayan M, Scheraga HA (2000) Disulfide bonds and protein folding. Biochemistry 39(15):4207–4216
Willner D, Trail PA, Hofstead SJ, King HD, Lasch SJ, Braslawsky GR, Greenfield RS, Kaneko T, Firestone RA (1993) (6-maleimidocaproyl)hydrazone of doxorubicin – a new derivative for the preparation of immunoconjugates of doxorubicin. Bioconjug Chem 4(6):521–527
Wong SS (1991) Chemistry of protein conjugation and cross-linking. CRC Press, Boca Raton, FL, p 340
Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, Forero-Torres A (2010) Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med 363(19):1812–1821
Zimmerman ES, Heibeck TH, Gill A, Li X, Murray CJ, Madlansacay MR, Tran C, Uter NT, Yin G, Rivers PJ, Yam AY, Wang WD, Steiner AR, Bajad SU, Penta K, Yang W, Hallam TJ, Thanos CD, Sato AK (2014) Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem 25(2):351–361
Acknowledgements
We sincerely thank our Abzena colleagues, past and present, for their contributions to the development of the technology described. We also thank ImaginAb Inc. for kind use of Fig. 3.10. Contact: martin.pabst@abzena.com or mark.frigerio@abzena.com
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Pabst, M., Bird, M., Frigerio, M., Godwin, A. (2017). Stable and Homogeneous Drug Conjugation by Sequential Bis-Alkylation at Disulphide Bonds Using Bis-Sulphone Reagents. In: Grawunder, U., Barth, S. (eds) Next Generation Antibody Drug Conjugates (ADCs) and Immunotoxins. Milestones in Drug Therapy. Springer, Cham. https://doi.org/10.1007/978-3-319-46877-8_3
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