Discovery of a potent, selective, and tumor-suppressing antibody antagonist of adenosine A2A receptor.

New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.

Effects of polymer end-group chemistry and order of deposition on controlled protein delivery from layer-by-layer assembly.

Layer-by-layer (LBL) assembly is an attractive platform for controlled release of biologics given its mild fabrication process and versatility in coating substrates of any shape. Proteins can be incorporated into LBL coatings by sequentially depositing oppositely charged polyelectrolytes, which self-assemble into nanoscale films on medical devices or tissue engineering scaffolds. However, previously reported LBL platforms often require the use of a few hundred layers to avoid burst release, which hinders their broad translation due to the lengthy fabrication process, cost, and batch-to-batch variability. Here we report a biodegradable LBL platform composed of only 10 layers with tunable protein release kinetics, which is an order of magnitude less than previously reported LBL platforms. We performed a combinatorial study to examine the effects of polymer chemistry and order of deposition of poly(ß-amino) esters on protein release kinetics under 81 LBL assembly conditions. Using the optimal "polyelectrolyte couples" for constructing the LBL film, basic fibroblast growth factor (bFGF) was released gradually over 14 days with retained biological activity to stimulate cell proliferation. The method reported herein is applicable for coating various substrates including metals, polymers, and ceramics and may be used for a broad range of biomedical and tissue engineering applications.

Discovery and design of G protein-coupled receptor targeting antibodies.

INTRODUCTION: G protein-coupled receptors (GPCRs) are the target of one-third of all approved drugs; however, these drugs only target about one-eighth of the human repertoire of GPCRs. GPCRs regulate a diverse range of critical physiological processes including organ development, cardiovascular function, mood, cognition, multicellularity, cellular motility, immune responses and sensation of light, taste, and odor. However, many GPCRs are expressed poorly, and a significant proportion have unknown ligands and unclear signaling pathways. AREAS COVERED: GPCRs are better suited to be targeted by monoclonal antibodies (mAbs) because of the challenges encountered in small-molecule discoveries such as druggability, selectivity, and distribution. mAbs have better drug-like properties in these respects. Herein, the authors review previously discovered functional mAbs that target GPCRs that are in the clinic and/or in development. They also review the biophysical considerations that make GPCRs so challenging to work with but also provide opportunities for biologic druggability. EXPERT OPINION: GPCRs are proven targets of small molecules yet remain an under-represented target of biologics. We believe that antibody drugs that target GPCRs have the potential to unlock new therapeutic avenues and also uncover previously unappreciated receptor biology, particularly when harnessing next-generation biologic modalities.

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity.

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

Mammalian synthetic biology for studying the cell.

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology.