This approach allowed an anti-HER2 sdAb homodimerCDBCO to be conjugated with an azido-CD3 sdAb. constructed from small binders targeting antigens of oncological importance, PD-L1 and HER2, were generated R-10015 with high purity, stability, and improved functionality compared to monomeric building blocks. The modularity of the approach was demonstrated through elaboration of the linker moiety through a bioorthogonal propargyl handle to produce proteinCproteinCfluorophore conjugates. Furthermore, extending the functionality of the homobifunctional reagents by temporarily masking reactive thiols included in R-10015 the linker allowed the assembly of higher order trimeric and tetrameric single-domain antibody conjugates. The potential for the approach to R-10015 be extended to proteins of greater biochemical complexity was demonstrated in the production of immunoglobulin single-domain antibody conjugates. On-demand control of thio-succinimide hydrolysis R-10015 combined with the facile assembly of chemically defined homo- and heterodimers constitutes an important expansion of the chemical methods available for generating stable proteinCprotein conjugates. Introduction ProteinCprotein conjugates stand as a unique class of biomolecules that combine two native proteins into one single scaffold, unlocking novel modes of actions with increasing impact in biotechnology and biopharmaceutical research and development.1,2 Applications include the generation of bifunctional engineered enzymes, antibodyCenzyme conjugates, immunotoxins, immunocytokines, bispecific antibodies, and imaging, using fluorescent protein fusions.3?10 Traditionally, these proteinCprotein conjugates have been derived from the recombinant expression of fusion proteins.1,11?13 Although this represents an indispensable strategy, there remain several key drawbacks. These include the restrictive requirement for N-to-C terminal ligation, potential for incorrect protein folding, poor expression yields, and incompatibility of constituent protein expression systems, thus prohibiting coexpression.7,11,13 Post-translational proteinCprotein conjugation offers an alternative strategy in which constituent proteins are independently expressed prior to post-translational ligation. Expression followed by subsequent conjugation at preselected amino acid residues obviates the requirement for N-to-C terminal conjugates, allowing greater topological diversity to be explored.14,15 Furthermore, the ability to produce incompatible constituent proteins in separate expression hosts gives the potential to create proteinCprotein conjugates that are inaccessible in the form of a recombinantly expressed fusion protein.7 Examples of post-translational approaches include enzymatic and tag-based methods, the incorporation of noncanonical amino acids with bioorthogonal reactivity profiles, as well as heterobifunctional and homobifunctional chemical linking strategies.16?19 The TM4SF2 latter represents a popular approach due to the inherent simplicity of linker synthesis and its application in the production of proteinCprotein conjugates. Cysteine residues represent one of the most frequently targeted canonical amino acids in site-selective bioconjugation.20?22 This popularity can be attributed to the low abundance of cysteine residues in the proteome (<2%),23 further limited by many being unavailable for conjugation due to being involved in disulfide bonding,24 coupled with the inherent nucleophilicity of the thiolate group. The site-selective Michael addition reaction of cysteine residues with maleimide reagents remains the most reliable reaction when producing proteinCsmall molecule conjugates.25 Naturally, the popularity of the cysteineCmaleimide reaction holds true when considering homobifunctional reagents in the context of chemically mediated proteinCprotein conjugation, in the form of bis-maleimide reagents (Figure ?Figure11).26?29 The high second order rate constants (k2 = 102 C 104 MC1 sC1), relative to other common cysteine modifying reagents, helps to overcome the proteinCprotein coupling problem.30?32 In brief, the proteinCprotein coupling problem relates to the challenge associated with ligating two sterically encumbered coupling partners at low concentrations typically associated with reactions involving biomolecules (usually below 100 M).19 Biorthogonal reactions such as copper-catalyzed azideCalkyne cycloaddition (CuAAC) and inverse electron-demand DielsCAlder reaction (IEDDA) have successfully been utilized in the preparation of proteinCprotein conjugates due to their favorable reaction rates.19 However, the requirement for installation of biorthogonal handles onto protein monomers adds additional steps, making these approaches more cumbersome and less attractive than direct conjugation through cysteine residues via a homobifunctional linking strategy. Open in a separate window Figure 1 Overview of maleimide-based homobifunctional linker strategies in proteinCprotein conjugation. Although maleimides offer an attractive reactivity profile, their utility comes with an important caveat. The retro-Michael deconjugation of maleimides and subsequent trapping by endogenous thiols leads to degradation of the resulting conjugate, and first generation maleimides do not represent a suitable approach for producing stable proteinCprotein conjugates (Figure ?Figure11).25 Various cysteine-based proteinCprotein conjugation technologies have been devised to overcome this issue, although mostly at the cost of slower kinetics compared to maleimideCcysteine conjugation. These include cysteine alkynylation using bis-5-(alkynyl)dibenzothiophenium triflate reagents,33 cysteine arylation using homobifunctional perfluoroaromatic reagents,34 and palladium-mediated proteinCprotein cross coupling.35 It is also known that ring-opening hydrolysis of the thio-succinimide.