It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody–drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C–C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

With the development of covalent modification strategies for viral capsids comes the ability to convert them into modular carrier systems for drug molecules and imaging agents. With this overall goal in mind, we have used two orthogonal modification strategies to decorate the exterior surface of genome-free MS2 capsids with PEG chains, while installing 50-70 copies of a fluorescent dye inside as a drug cargo mimic. Despite the very high levels of modification, the capsids remained in the assembled state, as determined by TEM, size-exclusion chromatography, and dynamic light scattering analysis. The ability of the polymer coating to block the access of polyclonal antibodies to the capsid surface was probed using a sandwich ELISA, which indicated a 90% reduction in binding. Further experiments indicated that biotin groups placed at the distal ends of the polymer chains were still capable of binding to streptavidin, despite their proximity to the PEG layer. Finally, a modular strategy was developed for the attachment of small-molecule targeting groups to the polymer chains through an efficient oxime formation reaction. As a result of these studies, a robust and versatile new platform has emerged for the potential delivery of therapeutic cargo.

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ( Re tricarbonyl 1,10-phenanthroline methylpyridyl rhenium I ). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl) pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state 3 Re is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of protoncoupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.proton-coupled electron transfer | radical generation | radical transport T he initiation and transport of many amino acid radicals occurs by proton-coupled electron transfer (PCET) (1-4). Accordingly, the importance of radicals in biological function (5) provides an imperative for the description of PCET in natural systems whose functions derive from radical-based chemistry (6). The PCET activity of amino acid radicals originates from the dependence of the reduction potential on the pK a of the amino acid in its oxidized and reduced states as well as the intrinsic redox potentials of the amino acid in its protonated and deprotonated states, and its association with hydrogen bonded partners as has been shown in proteins (7,8), β-hairpin peptides with interstrand dipolar contacts (9,10), and other de novo designed protein maquettes (11).Ribonucleotide reductases (RNRs) are essential enzymes of all organisms (12) that demonstrate exquisite control of radical transport for their function (13). RNRs catalyze the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs) and are therefore largely responsible for maintaining the cellular pool of monomeric DNA precursors. E. coli class Ia RNR consists of two homodimeric subunits, α2 and β2 (14,15). The α2 subunit contains the enzyme active site as well as two allosteric regulation sites, whereas β2 contains a diferric tyrosyl cofactor (•Y122), which is where the radical resides in the resting state of RNR. Nucleoside reduction requires formation of an α2∶β2 complex. Substrate turnover occurs by a radical mechanism mediated by an active site cysteine thiyl radical in α2 (•C439). A docking model (Fig. S1) based on crystal structure...

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 A, which occurs by a multi-step proton-coupled electron transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface—Y356F(β) and Y731F(α)—this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)–Y731(α)–Y730(α) triad is crucial to radical transport in RNR.