Stability of Maleimide-Based Antibody Drug Conjugates (ADCs)
There are a number of challenges in the design of ADCs with stability during circulation (1).  Wei and coworkers at Pfizer have a recent publication in Analytical Chemistry describing the transfer of a drug(Auristatin) from an ADC to a plasma protein (2).   These investigators observed that a greater amount of  drug (payload) was “lost” from the ADC than could be explained by the presence of free drug.  Further investigation revealed that the “missing” drug could be accounted for by transfer to albumin, most likely to cysteine 34.  Auristatin is linked to an antibody, trastuzumab, which is intended to carry the drug to the target cell; in this case the HER2 protein on tumor cells (3).  The linker consists of a protease-sensitive peptide sequence containing a terminal maleimide function. The maleimide reaction with a cysteine residues in the monoclonal antibody forming a thioether bond which has been considered to be stable (4). It should be mentioned that previous thinking on the stability of the thioether bond is based on loss-of-activity from a modified enzyme or by the isolation of a peptide containing a modified cysteine residue.  The stability of an ADC over an extended period of time in blood is subject to challenges such the presence of thiols such as cysteine or glutathione which are not seen in classical protein chemistry.  As a matter of history, the lability of the N-ethylmaleimide modification of glutathione was noted by Ernest Beutler and coworkers in 1970 (5).  These investigators also reported that the reversal of the N-ethylmaleimide modification was not catalyzed by an enzyme.  In later work, the reversibility of N-ethylmaleimide modification of thiols was observed with electrolysis (6).
The formation of the thioether bond with maleimide chemistry is a Michael addition where an electron-rich compounds such as a thiolate anion serves a Michael Donor in reacting with an α,β-unsaturated compound such as N-ethylmaleimide which is a Michael Acceptor.  The reversal of the thioether bond is referred to as a retro-Michael-type addition or reaction (7).  Since the equilibrium in the Michael addition reactions with a thiol as the Michael Donor is toward the formation of the adduct, glutathione is usually included in retro-Michael-type additions to measure the reaction (8).  While the lability of the ADC described by Wei and coworkers (2) was not a surprise, the transfer of the “payload” to the thiol group in albumin (Cys34) was a surprise since glutathione is more reactive with a Michael acceptor (acrylamide) than Cys34 in albumin (9).  The transfer of drug from an ADC to Cyy34 in albumin has been reported by other workers (10)
 Both the structure of the Michael acceptor and Michael donor affect the stability of a Michael adduct.   Zheng and coworkers (11) showed that monocyclic cyanoenones (Figure 1) were very reactive with Michael donors such as dithiothreitol and that reaction was reversible.  Serafimova and workers (12) observed that the adduct formed with an acrylate and 2-mercaptoethanol was stable, the adduct formed with 2-mercaptoethanol and cyanoacrylate (Figure 1) was reversible.   Similar reversibility was observed with acrylonitriles with substituents such as cyanophenyl or pyridyl (Figure 1) (13) or some benzalcyanoacetamides (Figure 1) (14).   The structure of the Michael donor also has an influence on the stability of a Michael conjugate as shown by Baldwin and Kiick (8).  The half-life of an adduct formed between NEM and 4-mercaptophenylacetic acid was 19 hrs while the half-life an adduct formed between NEM and N-acetylcysteine was 337 hrs.  Michael adducts with thiols may be stabilized by opening of the maleimide ring by hydrolysis(Figure 2) under (usually)basic conditions (15,16) as originally demonstrated in the development of a diagonal process for isolating cysteine-containing peptides (17). This hydrolytic reaction yields a N-ethylsuccinamic acid from the N-ethylsuccinimide products.  A self-hydrolyzing maleimide for ADC has been reported (18).  Shimmi and coworkers(10) have reported hydrolysis of maleimide on an anion-exchange column.
While involved in this work, I found some interesting material on the Michael reaction. First, Zheng and coworkers (11) in their elegant work on the cyanoenones obtained data suggested that chloride ion could function as a Michael acceptor. These investigators noted that the concentration of chloride in the reaction mixture was 1540 times that of the dithiothreitol.   Further investigation on my part found that NEM forms a stable adduct with sulfide (19,20).
1. Gordon, M.R., Canakci, M., Li, L., et al., Field guide to challenges and opportunities in antibody-drug conjugates for chemists, Bioconjug.Chem. 26, 2198-2215, 2015
2.  Wei, C., Zheng, G., Chark, T., et al., 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. 88, 4979-4986, 2016
3.  Baselga, J., Clinical trials of single-agent trastuzumab: (Herceptin), Semin.Oncol. 27(5 Suppl 9), 20-26, 2000
4. Lundblad, R.L., Modification of Proteins with Chemical Reagents, 4th edn., CRC/Taylor & Francis, Boca Raton, Florida, USA, 2014
5.  Beutler, E., Srivastava, S.K., and West, C., The reversibility of N-ethylmaleimide (NEM) alkylation of a red cell glutathione
6.  NIshiyama, J. and Kuninori, T., Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide  alkylation of thiols combined with electrolysis, Anal.Biochem. 200, 230-234, 1992
7. Fritz, K.S., Kellersberger, K.A., Gomez, J.D., and Petersen, D.R., 4-HNE adduct stability characterized by collision-induced dissociation and electron transfer dissociation mass spectrometry, Chem.Res.Toxicol. 25, 965-970, 2012
8.  Baldwin, A.D. and Kiick, K.L., Tunable degradation of maleimide thiol adducts in reducing environments, Bioconjug.Chem. 22, 1945-1953, 2011
9.  Tong, G.C., Corwall, W.K., and Means, G.K., Reaction of acrylamide with glutathione and serum albumin, Toxiol.Lett. 147, 127-131, 2004
10.  Shimmi, D., Taguchi, E., Iwano, J., et al., One-step conjugation method for site-specific antibody-drug conjugates through reactive cysteine -engineered antibody, Bioconug.Chem. 27, 1324-1336. 3026
11.  Zheng, S., Laxmi, Y.R.S., David, E., et al., Synthesis, chemical reactivity as Michael acceptors, and biological potency of monocyclic cyanoenones. Novel and highly potent anti-inflammatory and cytoprotective agents, J.Med.Chem. 55, 4837-4846, 2012
12.  Serafimova, I.M., Pufall, M.A., Krishnam, S., et al., Reversible targeting of noncatatalytic cysteines with chemically tuned electrophiles, Nat.Chem.Biol. 8, 471-476, 2012
13.  Krishnan, S., Miller, R.M., Tian, B., et al., Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis, J.Am.Chem.Soc. 136, 12624-12630, 2014
14.  Zheng, Y., Xu, Y., and Anslyn, E.V., Studies of reversible conjugate additions, Eur.J.Org.Chem. 2013, 5617-5621, 2013
15. Weissman, M.R., Winger, K.T., Ghassian, S., Gobbo, P. and Workentin, M.S., Insights into the application of the retro-Michael type addition on Maleimide-functionalized gold nanoparticles in biology and nanomedicine, Bioconjug.Chem. 27, 586-593, 2015
16. Fontaine, S.D., Reid, R., Robinson, L., Ashley, G.W., and Santi, D.V., Long-term stabilization of maleimide-thiol conjugates, Bioconjug.Chem. 26, 145-152, 2015
17.  Gehring, H. and Christen, P., A diagonal procedure for isolating sulfhydryl peptides alkylated with N-ethylmaleimide, Anal.Biochem. 107, 358-361, 1980
18. Lyon, R.P., Setter, J.R., Boyce, T.D., et al., Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates, Nat.Biotech. 37, 1059-1069, 2014
19.  Ellis, R.J., A rapid assay for sulfite reductase, Biochim.Biophys.Acta 85, 335-338, 1964
20.  Nashef, A.S., Osuga, D.T., and Feeney,R.C., Determination of hydrogen sulfide with 5,5’-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, and parachloromercuribenzoate, Anal.Biochem. 79, 394-405, 1977