Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

A bioinspired scaffold for rapid oxygenation of cell encapsulation systems.

Inadequate oxygenation is a major challenge in cell encapsulation, a therapy which holds potential to treat many diseases including type I diabetes. In such systems, cellular oxygen (O2) delivery is limited to slow passive diffusion from transplantation sites through the poorly O2-soluble encapsulating matrix, usually a hydrogel. This constrains the maximum permitted distance between the encapsulated cells and host site to within a few hundred micrometers to ensure cellular function. Inspired by the natural gas-phase tracheal O2 delivery system of insects, we present herein the design of a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O2 diffusivity than hydrogels. We incorporate the scaffold into a bulk hydrogel containing cells, which facilitates rapid O2 transport through the whole system to cells several millimeters away from the device-host boundary. A computational model, validated by in vitro analysis, predicts that cells and islets maintain high viability even in a thick (6.6 mm) device. Finally, the therapeutic potential of the device is demonstrated through the correction of diabetes in immunocompetent mice using rat islets for over 6 months.

Developing mechanically robust, triazole-zwitterionic hydrogels to mitigate foreign body response (FBR) for islet encapsulation.

Zwitterionic hydrogels such as those based on polycarboxybetaine (PCB) or polysulfobetaine (PSB) have potential for various biomedical applications, due to their biocompatibility and low biofouling properties. However, the poor mechanical properties of zwitterionic hydrogels developed to date remain a challenge, severely limiting their practical uses. To improve the mechanical properties without compromising their zwitterionic feature or biocompatibility, we designed a new class of zwitterionic hydrogels by introducing triazole moieties into the hydrogel monomers that could form energy-dissipating π-π stacking. Compared to conventional zwitterionic hydrogels, the triazole-zwitterionic (TR-ZW) ones exhibited similarly excellent antifouling properties, but were much more mechanically robust with higher stretchability (250% tensile strain), better compression-resistance (89% compressive strain and 65% compression for at least 10 cycles without any crack) and better folding-resistance. In addition, upon subcutaneous implantation in mice, the TR-ZW hydrogels induced significantly lower foreign body responses (FBR) (i.e. less fibrosis and more blood vessel formation relative to a poly(2-hydroxyethyl methacrylate) hydrogel control). As an example of their potential applications, we showed the use of the TR-ZW hydrogels for islet encapsulation and transplantation and demonstrated diabetes correction up to ~1 month in mice in the convenient subcutaneous site.

An Atmosphere-Breathing Refillable Biphasic Device for Cell Replacement Therapy.

Cell replacement therapy is emerging as a promising treatment platform for many endocrine disorders and hormone deficiency diseases. The survival of cells within delivery devices is, however, often limited due to low oxygen levels in common transplantation sites. Additionally, replacing implanted devices at the end of the graft lifetime is often unfeasible and, where possible, generally requires invasive surgical procedures. Here, the design and testing of a modular transcutaneous biphasic (BP) cell delivery device that provides enhanced and unlimited oxygen supply by direct contact with the atmosphere is presented. Critically, the cell delivery unit is demountable from the fixed components of the device, allowing for surgery-free refilling of the therapeutic cells. Mass transfer studies show significantly improved performance of the BP device in comparison to subcutaneous controls. The device is also tested for islet encapsulation in an immunocompetent diabetes rodent model. Robust cell survival and diabetes correction is observed following a rat-to-mouse xenograft. Lastly, nonsurgical cell refilling is demonstrated in dogs. These studies show the feasibility of this novel device for cell replacement therapies.

Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation.

Foreign body reaction (FBR) to implanted biomaterials and medical devices is common and can compromise the function of implants or cause complications. For example, in cell encapsulation, cellular overgrowth (CO) and fibrosis around the cellular constructs can reduce the mass transfer of oxygen, nutrients and metabolic wastes, undermining cell function and leading to transplant failure. Therefore, materials that mitigate FBR or CO will have broad applications in biomedicine. Here we report a group of zwitterionic, sulfobetaine (SB) and carboxybetaine (CB) modifications of alginates that reproducibly mitigate the CO of implanted alginate microcapsules in mice, dogs and pigs. Using the modified alginates (SB-alginates), we also demonstrate improved outcome of islet encapsulation in a chemically-induced diabetic mouse model. These zwitterion-modified alginates may contribute to the development of cell encapsulation therapies for type 1 diabetes and other hormone-deficient diseases.

Engineering transferrable microvascular meshes for subcutaneous islet transplantation.

The success of engineered cell or tissue implants is dependent on vascular regeneration to meet adequate metabolic requirements. However, development of a broadly applicable strategy for stable and functional vascularization has remained challenging. We report here highly organized and resilient microvascular meshes fabricated through a controllable anchored self-assembly method. The microvascular meshes are scalable to centimeters, almost free of defects and transferrable to diverse substrates, ready for transplantation. They promote formation of functional blood vessels, with a density as high as ~220 vessels mm-2, in the poorly vascularized subcutaneous space of SCID-Beige mice. We further demonstrate the feasibility of fabricating microvascular meshes from human induced pluripotent stem cell-derived endothelial cells, opening a way to engineer patient-specific microvasculature. As a proof-of-concept for type 1 diabetes treatment, we combine microvascular meshes and subcutaneously transplanted rat islets and achieve correction of chemically induced diabetes in SCID-Beige mice for 3 months.

Physical confinement induces malignant transformation in mammary epithelial cells.

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

Toll-like receptors TLR2 and TLR4 block the replication of pancreatic ß cells in diet-induced obesity.

Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.

Dynamic DNA material with emergent locomotion behavior powered by artificial metabolism.

Metabolism is a key process that makes life alive-the combination of anabolism and catabolism sustains life by a continuous flux of matter and energy. In other words, the materials comprising life are synthesized, assembled, dissipated, and decomposed autonomously in a controlled, hierarchical manner using biological processes. Although some biological approaches for creating dynamic materials have been reported, the construction of such materials by mimicking metabolism from scratch based on bioengineering has not yet been achieved. Various chemical approaches, especially dissipative assemblies, allow the construction of dynamic materials in a synthetic fashion, analogous to part of metabolism. Inspired by these approaches, here, we report a bottom-up construction of dynamic biomaterials powered by artificial metabolism, representing a combination of irreversible biosynthesis and dissipative assembly processes. An emergent locomotion behavior resembling a slime mold was programmed with this material by using an abstract design model similar to mechanical systems. Dynamic properties, such as autonomous pattern generation and continuous polarized regeneration, enabled locomotion along the designated tracks against a constant flow. Furthermore, an emergent racing behavior of two locomotive bodies was achieved by expanding the program. Other applications, including pathogen detection and hybrid nanomaterials, illustrated further potential use of this material. Dynamic biomaterials powered by artificial metabolism could provide a previously unexplored route to realize "artificial" biological systems with regenerating and self-sustaining characteristics.

Cu(II)/Proline-Catalyzed Reductive Coupling of Sulfuryl Chloride and P(O)-H for P-S-C Bond Formation.

A considerably improved method for the Cu-catalyzed coupling of sulfuryl chloride with P(O)-H was described. Using commercially available l-proline as the ligand decreased the precatalyst loading, broadened the substrate scope and greatly promoted the efficiency of the coupling reaction. Moreover, gram-scale preparation, easy-to handle and recyclable catalyst featured this transformation.

Designing a retrievable and scalable cell encapsulation device for potential treatment of type 1 diabetes.

Cell encapsulation has been shown to hold promise for effective, long-term treatment of type 1 diabetes (T1D). However, challenges remain for its clinical applications. For example, there is an unmet need for an encapsulation system that is capable of delivering sufficient cell mass while still allowing convenient retrieval or replacement. Here, we report a simple cell encapsulation design that is readily scalable and conveniently retrievable. The key to this design was to engineer a highly wettable, Ca2+-releasing nanoporous polymer thread that promoted uniform in situ cross-linking and strong adhesion of a thin layer of alginate hydrogel around the thread. The device provided immunoprotection of rat islets in immunocompetent C57BL/6 mice in a short-term (1-mo) study, similar to neat alginate fibers. However, the mechanical property of the device, critical for handling and retrieval, was much more robust than the neat alginate fibers due to the reinforcement of the central thread. It also had facile mass transfer due to the short diffusion distance. We demonstrated the therapeutic potential of the device through the correction of chemically induced diabetes in C57BL/6 mice using rat islets for 3 mo as well as in immunodeficient SCID-Beige mice using human islets for 4 mo. We further showed, as a proof of concept, the scalability and retrievability in dogs. After 1 mo of implantation in dogs, the device could be rapidly retrieved through a minimally invasive laparoscopic procedure. This encapsulation device may contribute to a cellular therapy for T1D because of its retrievability and scale-up potential.

Phase-Selective Syntheses of Cobalt Telluride Nanofleeces for Efficient Oxygen Evolution Catalysts.

Cobalt-based nanomaterials have been intensively explored as promising noble-metal-free oxygen evolution reaction (OER) electrocatalysts. Herein, we report phase-selective syntheses of novel hierarchical CoTe2 and CoTe nanofleeces for efficient OER catalysts. The CoTe2 nanofleeces exhibited excellent electrocatalytic activity and stablity for OER in alkaline media. The CoTe2 catalyst exhibited superior OER activity compared to the CoTe catalyst, which is comparable to the state-of-the-art RuO2 catalyst. Density functional theory calculations showed that the binding strength and lateral interaction of the reaction intermediates on CoTe2 and CoTe are essential for determining the overpotential required under different conditions. This study provides valuable insights for the rational design of noble-metal-free OER catalysts with high performance and low cost by use of Co-based chalcogenides.

High-water-content and resilient PEG-containing hydrogels with low fibrotic response.

Hydrogels such as those based on polyethylene glycol (PEG) are broadly used in biomedicine where high water contents, robust mechanical properties such as resilience and favorable interactions with the body are often simultaneously desirable. However, the mechanical properties of conventional hydrogels often degrade rapidly after swelling or with increasing water content, limiting their potential in many applications. Here we describe a new class of PEG-containing hydrogels that remain highly resilient after maximum swelling. We achieved the hydrogels by incorporating reversible "dual" hydrogen bonding into highly coiled, elastic PEG networks. These hydrogels, due to their high water content and high mechanical resilience, can form highly permeable, yet durable and easy-to-handle cell delivery devices without any additional structural support. In addition, optimization of chemical composition resulted in hydrogels with superior bio-inertness, inducing much less fibrosis upon subcutaneous implantation in mice than a polyhydroxyethylmethacrylate (PHEMA) hydrogel control. STATEMENT OF SIGNIFICANCE: Hydrogels such as polyethylene glycol (PEG)-based ones are broadly used in the biomedical world. Examples include wound dressings, tissue scaffolds, medical implants, biosensors and drug or cell delivery devices. In many of these applications, robust mechanical property, high water content (or facile mass transfer) and favorable interactions with the body are often simultaneously desirable. However, the mechanical property of hydrogels often degrades rapidly after swelling or with increasing water content. Here we report a new class of PEG-based hydrogels that simultaneously possess high water content, high mechanical resilience and low fibrotic response upon subcutaneous implantation in mice. These hydrogels may therefore find broad applications in biomedicine.

Scalable Production and Cryostorage of Organoids Using Core-Shell Decoupled Hydrogel Capsules.

Organoids, organ-mimicking multicellular structures derived from pluripotent stem cells or organ progenitors, have recently emerged as an important system for both studies of stem cell biology and development of potential therapeutics; however, a large-scale culture of organoids and cryopreservation for whole organoids, a prerequisite for their industrial and clinical applications, has remained a challenge. Current organoid culture systems relying on embedding the stem or progenitor cells in bulk extracellular matrix (ECM) hydrogels (e.g., Matrigel™) have limited surface area for mass transfer and are not suitable for large-scale productions. Here, we demonstrate a capsule-based, scalable organoid production and cryopreservation platform. The capsules have a core-shell structure where the core consists of Matrigel™ that supports the growth of organoids, and the alginate shell form robust spherical capsules, enabling suspension culture in stirred bioreactors. Compared with conventional, bulk ECM hydrogels, the capsules, which could be produced continuously by a two-fluidic electrostatic co-spraying method, provided better mass transfer through both diffusion and convection. The core-shell structure of the capsules also leads to better cell recovery after cryopreservation of organoids probably through prevention of intracellular ice formation.

Mass production of shaped particles through vortex ring freezing.

A vortex ring is a torus-shaped fluidic vortex. During its formation, the fluid experiences a rich variety of intriguing geometrical intermediates from spherical to toroidal. Here we show that these constantly changing intermediates can be 'frozen' at controlled time points into particles with various unusual and unprecedented shapes. These novel vortex ring-derived particles, are mass-produced by employing a simple and inexpensive electrospraying technique, with their sizes well controlled from hundreds of microns to millimetres. Guided further by theoretical analyses and a laminar multiphase fluid flow simulation, we show that this freezing approach is applicable to a broad range of materials from organic polysaccharides to inorganic nanoparticles. We demonstrate the unique advantages of these vortex ring-derived particles in several applications including cell encapsulation, three-dimensional cell culture, and cell-free protein production. Moreover, compartmentalization and ordered-structures composed of these novel particles are all achieved, creating opportunities to engineer more sophisticated hierarchical materials.

DNA Microgels as a Platform for Cell-Free Protein Expression and Display.

Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 µm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations.

Magnetic hydroxyapatite nanoworms for magnetic resonance diagnosis of acute hepatic injury.

Inorganic non-metallic biomaterials, including the silicon frustule of a unicellular diatom, the carbonate shell of a mollusk and the calcium skeleton of the vertebrate, which are the main constituent part of an organism, serve as the supportive and protective components of soft tissue. Among them, hydroxyapatite, which primarily makes up the enamel and bone, is widely used in tissue engineering. Recently, the inorganic nonmetallic biomaterials, especially the applications of hydroxyapatites have attracted great attention. Herein, we report a novel synthesis method of magnetic functionalized hydroxyapatite nanocomposites. By simply tuning the ratios of reactants, a series of hydroxyapatite-Fe3O4 worm-shaped nanocomposites (HAP-ION nanoworms) are obtained. In addition, layer-by-layer surface modifications with chitosan (CH) and sodium alginate (SA) were employed to improve the solubility and biocompatibility, and low cytotoxicity and no hemolysis were observed. With the increase of iron oxide nanocrystals, the magnetic properties of the magnetic assembled nanoworms were enhanced, which resulted in better performance of magnetic resonance (MR) imaging. Owing to the intravenous injection of HAP-ION nanoworms, the contrast to noise ratio (CNR) of hepatic MR imaging in vivo was enhanced obviously, which should be beneficial for hepatic injury grading and further therapeutic treatment.

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation.

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies.

Designing compartmentalized hydrogel microparticles for cell encapsulation and scalable 3D cell culture.

We describe here designs of compartmentalized hydrogel microparticles with a tunable extracellular matrix (ECM) support for cell encapsulation and scalable 3D cell culture. The microparticles, rapidly formed by a one-step, multi-fluidic electrostatic spraying technique (>10 000 min-1), have a uniform spherical shape, a nearly monodisperse size distribution and controlled compartmentalization. They not only have a high surface area for mass transfer but also offer defined space and essential ECM support for various scalable and efficient 3D cell culture, co-culture and microtissue production applications.