Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is a heterobifunctional amine-to-sulfhydryl crosslinker, which contains two reactive groups at opposite ends: N-hydroxysuccinimide-ester and maleimide, reactive with amines and thiols respectively. SMCC is often used in bioconjugation to link proteins with other functional entities (fluorescent dyes, tracers, nanoparticles, cytotoxic agents). For example, a targeted anticancer agent – trastuzumab emtansine (antibody-drug conjugate containing an antibody trastuzumab chemically linked to a highly potent drug DM-1) – is prepared using SMCC reagent. In a pharmacokinetics study, the CX-DM1-containing ADCs exhibited stability comparable to that of SMCC-DM1-containing ADCs. SMCC is a classic noncleavable linker that has been employed in Kadcyla. However, SMCC-based conjugates are still limited due to their instability in the circulatory system and hydrophobic properties.
Background References
1. Lambert J.M., Chari R.V. Ado-trastuzumab emtansine (T-DM1): an antibody-drug conjugate (ADC) for HER2− positive breast cancer. J Med Chem. 2014;57:6949–6964
2. Dovgan I., Kolodych S., Koniev O., Wagner A. 2-(Maleimidomethyl)-1,3-dioxanes (MD): a serum-stable self-hydrolysable hydrophilic alternative to classical maleimide conjugation. Sci Rep. 2016;6:30835
Indirect ELISA analysis of SMCC was performed by coating wells of a 96-well plate with 50 µL per well of different SMCC conjugations diluted in carbonate/bicarbonate buffer, at a concentration of 1 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 1%BSA blocking buffer for 1 hour at 37℃, and incubated with 50 µL per well of SMCC monoclonal antibody serial diluted starting from a concentration of 1µg/mL for 45 minutes at 37℃. The plate was washed and incubated with 50 µL per well of an HRP-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1/80,000 for 30 minutes at 37℃. Detection was performed using an Ultra TMB Substrate for 10 minutes at 37℃ in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.
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