Patrick G. Holder

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.

Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and elimination, and thus, there is a long clinical history associated with the use of recombinant cytokines to treat cancer. However, the use of cytokines as therapeutics has been limited by cytokine pleiotropy, complex biology, poor drug-like properties, and severe dose-limiting toxicities. Nevertheless, cytokines are crucial mediators of innate and adaptive antitumor immunity and have the potential to enhance immunotherapeutic approaches to treat cancer. Development of immune checkpoint inhibitors and combination immunotherapies has reinvigorated interest in cytokines as therapeutics, and a variety of engineering approaches are emerging to improve the safety and effectiveness of cytokine immunotherapy. In this review we highlight recent advances in cytokine biology and engineering for cancer immunotherapy.

We conduct a laboratory experiment in which participants can make donations to real charities. We vary whether the experimenter provides matching funds for any such donations, and whether there is individual or team competition for these matching funds. Our results indicate that providing matching funds for all donations does increase donations from 23% to 33% of the endowment. While individual competition for matching funds had nearly the same effectiveness as matching all donations, by far the most effective approach was to form (anonymous) teams that competed for matching funds; this led to donations of 47% of the endowment. We appeal to the notion of group identity to explain our results—participants seemed to be reluctant to “let down their team” in a competition. Our results can be seen as providing support for the notion that combining group identity and competition creates a motivation that can potentially be harnessed effectively for prosocial purposes. This paper was accepted by Uri Gneezy, behavioral economics.

To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.Background:Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo.Results:Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro.Conclusion:FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags.Significance:Understanding FGE biochemistry informs research on sulfatases and enables expanded biotechnology applications of the aldehyde tag. To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity. Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo. Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro. FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags.