Jonathan C. T. Carlson

Chemically controlled protein assembly: techniques and applications.

The study of biological processes has driven the efforts of modern molecular biology to unravel the microscopic capabilities of natural systems. Intrinsic to the experimental analysis of these life-governing principles is the process of testing, replicating, and visualizing the underlying biological mechanisms. As such, interacting with the nanoscale machinery of life becomes an increasingly apparent challenge.1 The range of this pursuit spans from DNA to RNA to proteins. While the controlled assembly of nucleic acid structures is widely studied,2,3 there are a smaller number of studies on the development of methods that investigate and exploit protein assembly and protein-protein interactions.4,5 These ubiquitous natural phenomena form a central founda-

BODIPY–Tetrazine Derivatives as Superbright Bioorthogonal Turn-on Probes†

The fastest and the brightest: A new design that intimately connects tetrazine to a BODIPY fluorophore enables exceptionally efficient energy transfer and quenching. Upon reaction of the tetrazine, the brightness of the fluorophore increases more than a thousand-fold, a fluorogenic activation up to two orders of magnitude greater than previously described.

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue-green region of the visible-light spectrum that display fluorescence enhancement exceeding 11,000-fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans-cyclooctene(TCO)-tetrazine chemistry platform. By exploiting highly efficient through-bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No-wash, fluorogenic imaging of diverse targets including cell-surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.

In vivo imaging reveals a tumor-associated macrophage–mediated resistance pathway in anti–PD-1 therapy

Tumor-associated macrophages limit anti–PD-1 effects by removing the antibody from CD8+ T cells. Tug-of-war with anti–PD-1 Antibodies against immune checkpoints such as programmed death–1 (PD-1) are gaining increasing prominence in cancer treatment, but even these promising therapeutics do not always work. To be effective in preventing T cells from becoming exhausted, anti–PD-1 antibodies must be able to remain bound to the T cells. Unfortunately, this does not always happen, as Arlauckas et al. discovered. Although anti–PD-1 antibodies initially bound to T cells as intended, the authors found that tumor-associated macrophages quickly removed these antibodies from T cells, thus inactivating them. The researchers also identified a potential way to overcome this problem, showing that inhibition of Fcγ receptors prevented removal of anti–PD-1 and prolonged its effects in vivo. Monoclonal antibodies (mAbs) targeting the immune checkpoint anti–programmed cell death protein 1 (aPD-1) have demonstrated impressive benefits for the treatment of some cancers; however, these drugs are not always effective, and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. We used in vivo imaging to uncover the fate and activity of aPD-1 mAbs in real time and at subcellular resolution in mice. We show that aPD-1 mAbs effectively bind PD-1+ tumor-infiltrating CD8+ T cells at early time points after administration. However, this engagement is transient, and aPD-1 mAbs are captured within minutes from the T cell surface by PD-1− tumor-associated macrophages. We further show that macrophage accrual of aPD-1 mAbs depends both on the drug’s Fc domain glycan and on Fcγ receptors (FcγRs) expressed by host myeloid cells and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcγRs before aPD-1 mAb administration substantially prolongs aPD-1 mAb binding to tumor-infiltrating CD8+ T cells and enhances immunotherapy-induced tumor regression in mice. These investigations yield insight into aPD-1 target engagement in vivo and identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy.

Bioorthogonal Fluorophore Linked DFO-Technology Enabling Facile Chelator Quantification and Multimodal Imaging of Antibodies.

Herein we describe the development and application of a bioorthogonal fluorogenic chelate linker that can be used for facile creation of labeled imaging agents. The chelate linker is based on the trans-cyclooctene(TCO)-tetrazine(Tz) chemistry platform and incorporates deferoxamine (DFO) as a (89)Zr PET tracer and a BODIPY fluorophore for multimodal imaging. The rapid (<3 min) ligation between mAb-TCO and Tz-BODIPY-DFO chelator is monitored using fluorescence and allows for determination of labeling completion. Utilizing BODIPY as the linker between mAb and DFO facilitates in chelator quantification using spectrophotometry, allowing for an alternative to traditional methods (mass and isotope dilution assay). Radiolabeling with (89)Zr to form (89)Zr-DFO-BODIPY-trastuzumab was found to be quantitative after incubation at room temperature for 1 h (1.5 mCi/mg specific activity). The cell binding assay using HER2+ (BT474) and HER2- (BT20) cell lines showed significant binding to (89)Zr-DFO-BODIPY-trastuzumab (6.45 ± 1.87% in BT474 versus 1.47 ± 0.39% in BT20). In vivo PET imaging of mice bearing BT20 or BT474 xenografts with (89)Zr-DFO-BODIPY-trastuzumab showed high tumor conspicuity, and biodistribution confirmed excellent, specific probe uptake of 237.3 ± 14.5% ID/g in BT474 xenografts compared to low, nonspecific probe uptake in BT20 xenografts (16.4 ± 5.6% ID/g) 96 h p.i. . Ex vivo fluorescence (465ex/520em) of selected tissues confirmed superb target localization and persistence of the fluorescence of (89)Zr-DFO-BODIPY-trastuzumab. The described platform is universally adaptable for simple antibody labeling.

Bioorthogonal Radiopaque Hydrogel for Endoscopic Delivery and Universal Tissue Marking

A novel dual marking hydrogel system is reported for radiological and laparoscopic localization of lesions. Bioorthogonally crosslinked hydrogel containing both tantalum and India ink can be rapidly formed inside the body after injecting precursors, and stably located for several days as a long-term biocompatible carrier for markers.

Unraveling tetrazine-triggered bioorthogonal elimination enables chemical tools for ultrafast release and universal cleavage

Recent developments in bond cleavage reactions have expanded the scope of bioorthogonal chemistry beyond click ligation and enabled new strategies for probe activation and therapeutic delivery. These applications, however, remain in their infancy, with further innovations needed to achieve the efficiency required for versatile and broadly useful tools in vivo. Among these chemistries, the tetrazine/trans-cyclooctene click-to-release reaction has exemplary kinetics and adaptability but achieves only partial release and is incompletely understood, which has limited its application. Investigating the mechanistic features of this reaction’s performance, we discovered profound pH sensitivity, exploited it with acid-functionalized tetrazines that both enhance and markedly accelerate release, and ultimately uncovered an unexpected dead-end isomer as the reason for poor release. Implementing facile methods to prevent formation of this dead end, we have achieved exceptional efficiency, with essentially complete release across the full scope of physiologic pH, potentiating drug-delivery strategies and expanding the dynamic range of bioorthogonal on/off control.

Single-cell barcode analysis provides a rapid readout of cellular signaling pathways in clinical specimens

Serial tissue sampling has become essential in guiding modern targeted and personalized cancer treatments. An alternative to image guided core biopsies are fine needle aspirates (FNA) that yield cells rather than tissues but are much better tolerated and have lower complication rates. The efficient pathway analysis of such cells in the clinic has been difficult, time consuming and costly. Here we develop an antibody-DNA barcoding approach where harvested cells can be rapidly re-stained through the use of custom designed oligonucleotide-fluorophore conjugates. We show that this approach can be used to interrogate drug-relevant pathways in scant clinical samples. Using the PI3K/PTEN/CDK4/6 pathways in breast cancer as an example, we demonstrate how analysis can be performed in tandem with trial enrollment and can evaluate downstream signaling following therapeutic inhibition. This approach should allow more widespread use of scant single cell material in clinical samples.Diagnostic tests need optimization to avoid invasive and costly repeated biopsies. Here the authors present an antibody-DNA barcoding approach where harvested single cells can be re-stained through custom oligonucleotide-fluorophore conjugates, enabling multiplexed analysis of cancer pathways.

Protein interface remodeling in a chemically induced protein dimer

Although the development of chemically induced, self‐assembled protein‐based materials is rapidly expanding, methods for directing their assembly in solution are sparse, and problems of population heterogeneity remain. By exerting control over the assembly of advanced protein structures, new classes of ordered protein nanomaterials become feasible, affecting numerous applications ranging from therapeutics to nanostructural engineering. Focusing on a protein‐based method for modulating the stability of a chemically induced dihydrofolate reductase (DHFR) dimer, we demonstrate the sensitivity of a methotrexate competition assay in determining the change in DHFR–DHFR binding cooperativity via interfacial mutations over a 1.3 kcal/mol range. This represents a change of more than 40% of the dimer complex binding energy conferred from protein–protein cooperativity (~3.1 kcal/mol). With the development of this investigative system and refinement of protein‐based techniques for complex stability modulation, the directed assembly of protein nanomaterials into heterocomplexes and a concomitant decrease in population heterogeneity becomes a realizable goal. Copyright