ABSTRACT
The use of monoclonal antibodies in cancer therapy is limited by their cross-reactivity to healthy tissue. Tumor targeting has been improved by generating masked antibodies that are selectively activated in the tumor microenvironment, but each such antibody necessitates a custom design. Here, we present a generalizable approach for masking the binding domains of antibodies with a heterodimeric coiled-coil domain that sterically occludes the complementarity-determining regions. On exposure to tumor-associated proteases, such as matrix metalloproteinases 2 and 9, the coiled-coil peptides are cleaved and antigen binding is restored. We test multiple coiled-coil formats and show that the optimized masking domain is broadly applicable to antibodies of interest. Our approach prevents anti-CD3-associated cytokine release in mice and substantially improves circulation half-life by protecting the antibody from an antigen sink. When applied to antibody-drug conjugates, our masked antibodies are preferentially unmasked at the tumor site and have increased anti-tumor efficacy compared with unmasked antibodies in mouse models of cancer.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Neoplasms/therapy , Animals , Antibodies, Monoclonal/chemistry , Cell Survival , Cytokines/metabolism , HEK293 Cells , Humans , Immunoconjugates , Integrins/metabolism , Mice , Models, Molecular , Protein Conformation , Protein DomainsABSTRACT
Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post-translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non-enzymatic methods to synthesize Ub chains of well-defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non-native linkages; however, few studies have examined the extent to which non-native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we compare the structure and function of Ub dimers bearing native and non-native linkages. Using small-angle X-ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non-native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady-state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non-native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non-native linkages can serve as surrogate substrates for explorations of Ub function.
Subject(s)
Ubiquitin/chemistry , Animals , Databases, Protein , Humans , Hydrolysis , Models, Molecular , Protein Conformation , Protein Multimerization , Scattering, Small Angle , Ubiquitin/metabolism , Ubiquitin-Specific Proteases/metabolism , X-Ray DiffractionABSTRACT
Covalent attachment of ubiquitin to target proteins is one of the most pervasive post-translational modifications in eukaryotes. Target proteins are often modified with polymeric ubiquitin chains of defined lengths and linkages that may further undergo dynamic changes in composition in response to cellular signals. Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We have discovered that a yeast ubiquitin C-terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site-specifically modified polyubiquitin chains produced by thiol-ene chemistry. We have used this chemoenzymatic approach to prepare dual-functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual-functionalized ubiquitin chains enabled the first real-time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy. In summary, this work provides a powerful new tool for elucidating the mechanisms of DUBs and other ubiquitin processing enzymes.
Subject(s)
Endopeptidases/metabolism , Ubiquitin-Specific Proteases/metabolism , Ubiquitin/metabolism , Biocatalysis , Endopeptidases/chemistry , Humans , Microscopy, Fluorescence , Ubiquitin/chemistry , Ubiquitin-Specific Proteases/chemistryABSTRACT
Linked: a method based on thiol-ene chemistry enables the synthesis and purification of ubiquitin oligomers with ≥4 units. This approach, which employs free-radical polymerization, can be applied towards the synthesis of homogeneous Lys6-linked ubiquitin oligomers currently inaccessible by enzymatic methods. By using these chains, one can study their roles in the ubiquitin proteasome system and the DNA damage response pathway.