Cytokine-based therapies for cancer have not achieved widespread clinical success because of inherent toxicities. Treatment for pancreatic cancer is limited by the dense stroma that surrounds tumors and by an immunosuppressive tumor microenvironment. To overcome these barriers, we developed constructs of single-domain antibodies (VHHs) against PD-L1 fused with IL-2 and IFNγ. Targeting cytokine delivery in this manner reduced pancreatic tumor burden by 50%, whereas cytokines fused to an irrelevant VHH, or blockade of PD-L1 alone, showed little effect. Targeted delivery of IL-2 increased the number of intratumoral CD8 T cells, whereas IFNγ reduced the number of CD11b cells and skewed intratumoral macrophages toward the display of M1-like characteristics. Imaging of fluorescent VHH-IFNγ constructs, as well as transcriptional profiling, demonstrated targeting of IFNγ to the tumor microenvironment. Many tumors and tumor-infiltrating myeloid cells express PD-L1, rendering them potentially susceptible to this form of targeted immunotherapy. .

No abstract

Fluorescence-based immunosensors serve a vital role in biotechnology and diagnostic and therapeutic applications. Our group recently developed a unique fluoroimmunosensor named Quenchbody (Q-body) that operates based on the principle of quenching and the antigen-dependent release of fluorophore, which is incorporated to a recombinant antibody fragment, either the single-chain Fv (scFv) or the Fab fragment of an antibody, using a cell-free transcription-translation system. With the objective of extending the functionality and diversity of the Q-body, here we attempted to make Q-bodies by labeling the recombinant scFv, which was prepared from E. coli using several commercially available dye-maleimides. As a result, we reproducibly obtained larger amounts of antiosteocalcin Q-bodies, with an improved yield and cost-efficiency compared with those obtained from a conventional cell-free system. The fluorescence intensity of each Q-body, including that labeled with newly tested rhodamine red, was significantly increased in the presence of an antigen with a low detection limit, although some differences in response were observed for the dye with different spacer lengths between dye and maleimide. The results indicate the Q-body’s applicability as a powerful multicolored sensor, with a potential to simultaneously monitor multiple targets in a sample.

Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions.

No abstract