Efficient and selective antibody modification with functionalised divinyltriazines†

Antibody–drug conjugates (ADCs) are a rapidly emerging class of therapeutics for the treatment of cancer. ADCs deliver potent cytotoxic agents to specific cells by exploiting the ability of the conjugated antibody to selectively target overexpressed cell-surface receptors. The success of the platform is attested by the upwards of 80 ADCs currently in clinical trials, and the eight ADCs that have been granted marketing approval by the Food and Drug Administration—the most recent being sacituzumab govitecan (Trodelvy®, Immunomedics) in April 2020 for the treatment of metastatic triple-negative breast cancer. Notwithstanding, many of the commonly adopted approaches to antibody conjugation have significant limitations. Common methods of attachment involve nucleophilic conjugation through multiple lysine or cysteine residues (generated by reduction of interchain disulfide bonds). Such stochastic modification strategies predominantly produce heterogeneous mixtures of conjugated antibody products, which accordingly suffer from unpredictable and inconsistent pharmacological profiles. Other approaches to attaining homogeneous constructs have been developed, such as the incorporation of engineered cysteine residues, unnatural amino acids, enzymatic modification, chemical linchpins, and affinity peptides—however may involve additional complexity. Site-selective disulfide rebridging has emerged as an alternative strategy towards the production of homogeneous ADCs. This method involves reduction of the interchain disulfides, followed by treatment with a linker which crosslinks the reactive thiols from the reduced disulfides, thus reforming the covalent bridge between the protein chains. Significant progress has been made in recent years in the development of disulfide rebridging linkers, with the toolbox of available reagents now including bissulfones, next generation maleimides, and pyridazinediones. Although these platforms constitute significant advances, new methodologies are required to further stimulate the development of homogeneous ADCs comprising native antibodies. A notable issue with disulfide rebridging strategies is the potential for the formation of non-native cysteine bridges, resulting in an undesirable mixture of conjugates. Moreover, many modification strategies typically require a large excess of conjugation reagent, which may affect development and production costs depending upon the strategy adopted for toxin incorporation. Accordingly, the development of protocols with improved stoichiometry is a necessity. In light of these challenges, we sought to develop a disulfide rebridging platform that proceeds with increased efficiency and economy. We have recently demonstrated that divinylpyrimidines (DVPs) can be used for the efficient rebridging of disulfide bonds in antibodies and antibody fragments, producing stable conjugates with consistent drug-to-antibody ratio (DAR). The ability of divinyltriazines (DVTs) to function as cysteine stapling reagents in the development of functionalised macrocyclic peptide therapeutics has also been showcased. It was hypothesised that these more electron deficient DVT reagents may enable an efficient and economical synthesis of ADCs that forms a product with increased homogeneity. As such, we elected to adopt triazine 1 as a simple and easily accessible scaffold upon which to centre our antibody conjugation investigations (Fig. 1). We began by examining the ability of model triazine 1 to rebridge the four interchain disulfide bonds found in an IgG1 antibody. Trastuzumab (Herceptin®) was selected for appraisal of the rebridging reaction. Commonly used in the evaluation †Electronic supplementary information (ESI) available. See DOI: 10.1039/ d0ob01002b Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, UK. E-mail: spring@ch.cam.ac.uk

Antibody-drug conjugates (ADCs) are a rapidly emerging class of therapeutics for the treatment of cancer. 1,2 ADCs deliver potent cytotoxic agents to specific cells by exploiting the ability of the conjugated antibody to selectively target overexpressed cell-surface receptors. The success of the platform is attested by the upwards of 80 ADCs currently in clinical trials, 1,3 and the eight ADCs that have been granted marketing approval by the Food and Drug Administration-the most recent being sacituzumab govitecan (Trodelvy®, Immunomedics) in April 2020 for the treatment of metastatic triple-negative breast cancer. Notwithstanding, many of the commonly adopted approaches to antibody conjugation have significant limitations. Common methods of attachment involve nucleophilic conjugation through multiple lysine or cysteine residues (generated by reduction of interchain disulfide bonds). Such stochastic modification strategies predominantly produce heterogeneous mixtures of conjugated antibody products, which accordingly suffer from unpredictable and inconsistent pharmacological profiles. [4][5][6] Other approaches to attaining homogeneous constructs have been developed, such as the incorporation of engineered cysteine residues, unnatural amino acids, enzymatic modification, chemical linchpins, and affinity peptides-however may involve additional complexity. [7][8][9][10] Site-selective disulfide rebridging has emerged as an alternative strategy towards the production of homogeneous ADCs. 11 This method involves reduction of the interchain disulfides, followed by treatment with a linker which crosslinks the reactive thiols from the reduced disulfides, thus reforming the covalent bridge between the protein chains. Significant progress has been made in recent years in the development of disulfide rebridging linkers, with the toolbox of available reagents now including bissulfones, 12 next generation maleimides, [13][14][15][16] and pyridazinediones. [17][18][19] Although these platforms constitute significant advances, new methodologies are required to further stimulate the development of homogeneous ADCs comprising native antibodies.
A notable issue with disulfide rebridging strategies is the potential for the formation of non-native cysteine bridges, resulting in an undesirable mixture of conjugates. 20 Moreover, many modification strategies typically require a large excess of conjugation reagent, which may affect development and production costs depending upon the strategy adopted for toxin incorporation. Accordingly, the development of protocols with improved stoichiometry is a necessity. [21][22][23] In light of these challenges, we sought to develop a disulfide rebridging platform that proceeds with increased efficiency and economy. We have recently demonstrated that divinylpyrimidines (DVPs) can be used for the efficient rebridging of disulfide bonds in antibodies and antibody fragments, producing stable conjugates with consistent drug-to-antibody ratio (DAR). 24,25 The ability of divinyltriazines (DVTs) to function as cysteine stapling reagents in the development of functionalised macrocyclic peptide therapeutics has also been showcased. 26 It was hypothesised that these more electron deficient DVT reagents may enable an efficient and economical synthesis of ADCs that forms a product with increased homogeneity. As such, we elected to adopt triazine 1 as a simple and easily accessible scaffold upon which to centre our antibody conjugation investigations (Fig. 1).
We began by examining the ability of model triazine 1 to rebridge the four interchain disulfide bonds found in an IgG 1 antibody. Trastuzumab (Herceptin®) was selected for appraisal of the rebridging reaction. Commonly used in the evaluation of antibody bioconjugation strategies, trastuzumab is a monoclonal IgG 1 antibody used in the treatment of HER2+ cancers, and is the antibody used in two approved ADCs (Kadcyla® and Enhertu®) and multiple ADCs currently undergoing clinical evaluation. 27 The four solvent accessible interchain disulfide bonds of trastuzumab were reduced in Tris buffer (25 mM Tris HCl, 25 mM NaCl, 0.5 mM EDTA; pH 8.0) with an excess of the reducing agent tris(2-carboxyethyl) phosphine hydrochloride (TCEP; 10 eq.), to reveal eight free thiols. Complete reduction was confirmed by LC-MS and SDS-PAGE analysis (ESI Fig. S1 †). The reduced antibody was then treated with DVT 1 (see ESI † for full synthetic details). Pleasingly, highly efficient rebridging of the disulfide bonds was observed with an excess of rebridging reagent (20 equivalents per IgG) over 4 hours at 37°C. The reaction was monitored by LC-MS and SDS-PAGE analysis, which evidenced the formation of the correctly rebridged 'full' antibody, alongside the 'half-antibody' species formed through intrachain bridging of the heavy chain hinge region cysteines (Fig. 2a). A recent study has suggested that although so called 'scrambling' of antibody disulfide bonds has a slight impact on the Fc profile, the critical biophysical properties (e.g. antigen binding, aggregation) of the conjugate are largely unaffected. 20 It is postulated that the scrambled conjugate remains effective as there is no change to the site of modification, the net DAR is unaffected, and the stability of the covalent linkage remains. Undesired 'half-antibody' formation has remained a persistent issue in the development of disulfide rebridging linkers, and so an optimisation study with triazine 1 was undertaken to determine conditions which favoured greater formation of the 'full' rebridged antibody conjugate. Sequential reduction of the antibody and linker addition across a range of conditions was undertaken, exploring the effect of varying the number of equivalents of linker, temperature, and duration of reaction (ESI Table S1 †). Reduced trastuzumab was incubated with 10, 20, 40, or 80 equivalents of triazine 1, each at temperatures of 4, 20 and 37°C. Each reaction was monitored over 8 hours. A number of observations were evident from this investigation. First, a significant dependence between temperature and the nature of bridging between the formed conjugates was found. Rebridging was found to occur with increased efficiency at higher temperatures (ESI Fig. S2 †). Pleasingly, treatment of the reduced antibody with just 10 molar equivalents of DVT 1 for 1 hour at 37°C was sufficient to afford excellent conversion to   rebridged antibody. Extending the reaction time beyond 1 hour, or treatment with triazine 1 in excess of 10 equivalents did not offer appreciable improvements in rebridging efficiency. It was also found that removing excess TCEP and its by-products prior to addition of the DVT reagent yielded a marginal improvement in the conversion efficiency of the reaction from unreacted light and heavy chain to the rebridged antibody (ESI Fig. S2 †). This suggested that TCEP or by-products produced as a result of it may slightly impede the rebridging reaction. Further investigation of the excess of triazine 1 required was then undertaken. Conjugations to trastuzumab were conducted with between 4-10 equivalents of triazine 1. Although minor increases in light and heavy chain presence was observed via SDS-PAGE with lower concentrations of DVT, the rate of rebridging was still remarkable given the stoichiometry (ESI Fig. S3 †). These results demonstrate that stoichiometric equivalents of triazine 1 can be used without significant detriment to the progression of the conjugation reaction.
Synthesis of antibody conjugate 5 allowed for the assessment of the plasma stability of DVT mediated bioconjugates. The fluorescent antibody conjugate 5 was incubated in reconstituted human plasma at 37°C for one week. Aliquots were taken throughout the incubation period and were analysed via SDS-PAGE; in-gel fluorescence and Coomassie staining revealed no significant transfer of the AlexaFluor™ 488 label to plasma proteins, or loss of antibody chain fragments (Fig. 3d). This result demonstrates that DVT linkers can be employed to generate stable bioconjugates.
Scheme 1 Amide coupling of PEG 2 -alkyne unit to form triazine 2. In conclusion, we have described the development of an efficient and economical strategy for the rebridging of antibody interchain disulfides. The linker equivalents required for a rapid production of plasma-stable antibody conjugates can be reduced to a near stoichiometric ratio, without significant detriment to reaction efficiency. The platform incorporates a vector that facilitates bioorthogonal divergent functionalisation (CuAAC). As such, the DVT scaffold provides a useful addition to the toolbox of reagents available for selective disulfide rebridging of proteins.

Conflicts of interest
There are no conflicts to declare.