Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.

Single-cell sorting based on secreted products for functionally defined cell therapies.

Cell therapies have emerged as a promising new class of "living" therapeutics over the last decade and have been particularly successful for treating hematological malignancies. Increasingly, cellular therapeutics are being developed with the aim of treating almost any disease, from solid tumors and autoimmune disorders to fibrosis, neurodegenerative disorders and even aging itself. However, their therapeutic potential has remained limited due to the fundamental differences in how molecular and cellular therapies function. While the structure of a molecular therapeutic is directly linked to biological function, cells with the same genetic blueprint can have vastly different functional properties (e.g., secretion, proliferation, cell killing, migration). Although there exists a vast array of analytical and preparative separation approaches for molecules, the functional differences among cells are exacerbated by a lack of functional potency-based sorting approaches. In this context, we describe the need for next-generation single-cell profiling microtechnologies that allow the direct evaluation and sorting of single cells based on functional properties, with a focus on secreted molecules, which are critical for the in vivo efficacy of current cell therapies. We first define three critical processes for single-cell secretion-based profiling technology: (1) partitioning individual cells into uniform compartments; (2) accumulating secretions and labeling via reporter molecules; and (3) measuring the signal associated with the reporter and, if sorting, triggering a sorting event based on these reporter signals. We summarize recent academic and commercial technologies for functional single-cell analysis in addition to sorting and industrial applications of these technologies. These approaches fall into three categories: microchamber, microfluidic droplet, and lab-on-a-particle technologies. Finally, we outline a number of unmet needs in terms of the discovery, design and manufacturing of cellular therapeutics and how the next generation of single-cell functional screening technologies could allow the realization of robust cellular therapeutics for all patients.

Wound healing with topical BRAF inhibitor therapy in a diabetic model suggests tissue regenerative effects.

Wound healing is a multi-step process to rapidly restore the barrier function. This process is often impaired in diabetic patients resulting in chronic wounds and amputation. We previously found that paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway via topical administration of the BRAF inhibitor vemurafenib accelerates wound healing by activating keratinocyte proliferation and reepithelialization pathways in healthy mice. Herein, we investigated whether this wound healing acceleration also occurs in impaired diabetic wounds and found that topical vemurafenib not only improves wound healing in a murine diabetic wound model but unexpectedly promotes hair follicle regeneration. Hair follicles expressing Sox-9 and K15 surrounded by CD34+ stroma were found in wounds of diabetic and non-diabetic mice, and their formation can be prevented by blocking downstream MEK signaling. Thus, topically applied BRAF inhibitors may accelerate wound healing, and promote the restoration of improved skin architecture in both normal and impaired wounds.

Accelerated wound healing by injectable star poly(ethylene glycol)-b-poly(propylene sulfide) scaffolds loaded with poorly water-soluble drugs.

Injectable hydrogel matrices take the shape of a wound cavity and serve as scaffold for tissue repair and regeneration. Yet these materials are generally hydrophilic, limiting the incorporation of poorly water soluble, hydrophobic drugs. Here we show this shortcoming is circumvented through a star-shaped amphiphilic block copolymer comprising poly(ethylene glycol) and poly (propylene sulfide). This star-shaped amphiphilic polymer self-assembles in an aqueous medium into a physically stable hydrogel and effectively dissolves hydrophobic molecules delivering them at therapeutic doses. The self assembled hydrogel is a robust three-dimensional scaffold in vivo effectively promoting cellular infiltration, reducing inflammation, and wound clsoure. When combined with a hydrophobic BRAF inhibitor that promotes paradoxical mitogen-activated protein kinase (MAPK) activation in keratinocytes and wound closure, our self assembled scaffold supported dermal wound closure at a reduced drug dosage compared to administering the drug in dimethyl sulfoxide (DMSO) without a polymeric matrix. This family of star-shaped amphiphilic polymers delivers poorly water soluble active agents at a fraction of generally required dosage for efficacy and supports three-dimensional cell growth at tissue wounds, showing great promise for novel uses of hydrophobic drugs in tissue repair applications.