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Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, leading to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia. Only a few reports describe methods for optimizing peptide linkers to maintain efficient and potent tumor payload delivery while enhancing circulating stability. Herein, we address these critical limitations through the development of a tandem-cleavage linker strategy, where two sequential enzymatic cleavage events mediate payload release. We prepared dipeptides that are protected from degradation in the circulation by a sterically-encumbering glucuronide moiety. Upon ADC internalization and lysosomal degradation, the monosaccharide is removed and the exposed dipeptide is degraded, liberating the attached payload inside the target cell. We used CD79b-targeted monomethyl auristatin E (MMAE) conjugates as our model system, and compared the stability, efficacy, and tolerability of ADCs made with tandem-cleavage linkers to ADCs made using standard technology with the vedotin linker. The results—where rat studies showed dramatically improved tolerability in the hematopoietic compartment—highlight the role that linker stability plays in efficacy and tolerability, and offer a means of improving an ADC’s therapeutic index for improved patient outcomes.

Antibody-drug conjugates (ADCs) are an established modality for the tissue-specific delivery of chemotherapeutics. However, due to the hydrophobic nature of many cytotoxic payloads, challenges remain in developing chemically stable ADCs with high drug loading. In previous studies, payload structure, unique stimuli-responsive chemistries, and PEGylated cross-linkers have been used to decrease ADC hydrophobicity. In this work, we investigate the effect of a new parameter, cross-linker sequence. A support-free synthesis of PEGylated, sequence-defined cross-linkers was developed and applied to the synthesis of three constitutionally isomeric ADCs containing PEG side chains and a monomethyl auristatin E payload. Placement of PEG side chains distally from the payload was found to yield an ADC with altered hydrophilicity, antigen binding, and in vitro potency. This work establishes a versatile method for synthesizing multifunctional cross-linkers and identifies cross-linker sequence as a new handle for modulating the performance of ADCs.

Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation-Michael addition. A pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional ligation reactions. Using this unique ligation, we generated three site-specifically labeled antibody-drug conjugates (ADCs) with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the site-specific ADCs are reported.

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.

Lipid-derived desiccation resistance in membranes is a rare, unique ability previously observed only with trehalose dimycolate (TDM), an abundant mycobacterial glycolipid. Here we present the first synthetic trehalose glycolipids capable of providing desiccation protection to membranes of which they are constituents. The synthetic glycolipids consist of a simple trehalose disaccharide headgroup, similar to TDM, with hydrophobic tail groups of two 15- or 18-carbon chains. The synthetic trehalose glycolipids protected supported monolayers of phospholipids against dehydration even as minority components of the overall membrane, down to as little as 20 mol % trehalose glycolipid as assessed by assays of membrane fluidity. The dependence of the desiccation protection on the synthetic trehalose glycolipid fraction is nearly identical to that of TDM. The striking similarity of the desiccation resistance observed with TDM and the synthetic trehalose glycolipids, despite the variety of hydrophobic tail structures employed, suggests that interactions between the trehalose headgroup and surrounding molecules are the determining factor in dehydration protection.

Fucosylated glycoproteins are involved in many cell-cell recognition events and are markers of embryonic and malignant tissue. Here we report a method for rapid profiling of fucosylated glycoproteins from human cells using 6-azido fucose as a metabolic label.

We developed a polymer coating for carbon nanotubes (CNTs) that mimics the mucin glycoprotein coating of mammalian cells. CNTs coated with these mucin mimic polymers have two novel properties: they can bind to carbohydrate receptors, providing a means for biomimetic interactions with cell surfaces, and, importantly, they are rendered nontoxic to cells.