Building safety into CAR-T therapy.

Chimeric antigen receptor T cell (CAR-T) therapy is an innovative immunotherapeutic approach that utilizes genetically modified T-cells to eliminate cancer cells using the specificity of a monoclonal antibody (mAb) coupled to the potent cytotoxicity of the T-lymphocyte. CAR-T therapy has yielded significant improvements in relapsed/refractory B-cell malignancies. Given these successes, CAR-T has quickly spread to other hematologic malignancies and is being increasingly explored in solid tumors. From early clinical applications to present day, CAR-T cell therapy has been accompanied by significant toxicities, namely cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and on-target off-tumor (OTOT) effects. While medical management has improved for CRS and ICANS, the ongoing threat of refractory symptoms and unanticipated idiosyncratic toxicities highlights the need for more powerful safety measures. This is particularly poignant as CAR T-cell therapy continues to expand into the solid tumor space, where the risk of unpredictable toxicities remains high. We will review CAR-T as an immunotherapeutic approach including emergence of unique toxicities throughout development. We will discuss known and novel strategies to mitigate these toxicities; additional safety challenges in the treatment of solid tumors, and how the inducible Caspase 9 "safety switch" provides an ideal platform for continued exploration.

The polymer and materials science of the bacterial fimbriae Caf1.

Fimbriae are long filamentous polymeric protein structures located upon the surface of bacteria. Often implicated in pathogenicity, the biosynthesis and function of fimbriae has been a productive topic of study for many decades. Evolutionary pressures have ensured that fimbriae possess unique structural and mechanical properties which are advantageous to bacteria. These properties are also difficult to engineer with well-known synthetic and natural fibres, and this has raised an intriguing question: can we exploit the unique properties of bacterial fimbriae in useful ways? Initial work has set out to explore this question by using Capsular antigen fragment 1 (Caf1), a fimbriae expressed naturally by Yersina pestis. These fibres have evolved to 'shield' the bacterium from the immune system of an infected host, and thus are rather bioinert in nature. Caf1 is, however, very amenable to structural mutagenesis which allows the incorporation of useful bioactive functions and the modulation of the fibre's mechanical properties. Its high-yielding recombinant synthesis also ensures plentiful quantities of polymer are available to drive development. These advantageous features make Caf1 an archetype for the development of new polymers and materials based upon bacterial fimbriae. Here, we cover recent advances in this new field, and look to future possibilities of this promising biopolymer.

Exploiting Meltable Protein Hydrogels to Encapsulate and Culture Cells in 3D.

There is a growing realization that 3D cell culture better mimics complex in vivo environments than 2D, lessening aberrant cellular behaviors and ultimately improving the outcomes of experiments. Chemically crosslinked hydrogels which imitate natural extracellular matrix (ECM) are proven cell culture platforms, but the encapsulation of cells within these hydrogel networks requires bioorthogonal crosslinking chemistries which can be cytotoxic, synthetically demanding, and costly. Capsular antigen fragment 1 (Caf1) is a bacterial, polymeric, fimbrial protein which can be genetically engineered to imitate ECM. Furthermore, it can, reversibly, thermally interconvert between its polymeric and monomeric forms even when chemically crosslinked within a hydrogel network. It is demonstrated that this meltable feature of Caf1 hydrogels can be utilized to encapsulate neonatal human dermal fibroblasts at a range of cell densities (2 × 105 -2 × 106  cells mL-1 of hydrogel) avoiding issues with chemical cytotoxicity. These hydrogels supported cell 3D culture for up to 21 d, successfully inducing cellular functions such as proliferation and migration. This work is significant because it further highlights the potential of simple, robust, Caf1-based hydrogels as a cell culture platform.

Unraveling the molecular determinants of the anti-phagocytic protein cloak of plague bacteria.

The pathogenic bacterium Yersina pestis is protected from macrophage engulfment by a capsule like antigen, F1, formed of long polymers of the monomer protein, Caf1. However, despite the importance of this pathogen, the mechanism of protection was not understood. Here we demonstrate how F1 protects the bacteria from phagocytosis. First, we show that Escherichia coli expressing F1 showed greatly reduced adherence to macrophages. Furthermore, the few cells that did adhere remained on the macrophage surface and were not engulfed. We then inserted, by mutation, an "RGDS" integrin binding motif into Caf1. This did not change the number of cells adhering to macrophages but increased the fraction of adherent cells that were engulfed. Therefore, F1 protects in two separate ways, reducing cell adhesion, possibly by acting as a polymer brush, and hiding innate receptor binding sites needed for engulfment. F1 is very robust and we show that E. coli expressing weakened mutant polymers are engulfed like the RGDS mutant. This suggests that innate attachment sites on the native cell surface are exposed if F1 is weakened. Single-molecule force spectroscopy (SMFS) experiments revealed that wild-type F1 displays a very high mechanical stability of 400 pN. However, the mechanical resistance of the destabilised mutants, that were fully engulfed, was only 20% weaker. By only marginally exceeding the mechanical force applied to the Caf1 polymer during phagocytosis it may be that the exceptional tensile strength evolved to resist the forces applied at this stage of engulfment.

Probing the oligomeric re-assembling of bacterial fimbriae in vitro: a small-angle X-ray scattering and analytical ultracentrifugation study.

Capsular antigen fragment 1 (Caf1) is an oligomeric protein consisting of 15 kDa monomeric subunits that are non-covalently linked through exceptionally strong and kinetically inert interactions into a linear polymer chain. It has been shown that after its thermal depolymerisation into unfolded monomeric subunits, Caf1 is able to efficiently repolymerise in vitro to reform its polymeric structure. However, little is known about the nature of the repolymerisation process. An improved understanding of this process will lead to the development of methods to better control the lengths of the repolymerised species, and ultimately, to better design of the properties of Caf1-based materials. Here we utilize small-angle X-ray scattering to estimate the size of Caf1 polymers during the first 24 h of the re-polymerisation process. Analytical ultracentrifugation measurements were also used to investigate the process post-24 h, where the rate of repolymerisation becomes considerably slower. Results show that in vitro polymerisation proceeds in a linear manner with no evidence observed for the formation of a lateral polymer network or uncontrolled aggregates. The rate of Caf1 in vitro repolymerisation was found to be concentration-dependent. Importantly, the rate of polymer growth was found to be relatively fast over the first few hours, before continuing at a dramatically slower rate. This observation is not consistent with the previously proposed step-growth mechanism of in vitro polymerisation of Caf1, where a linear increase in polymer length would be expected with time. We speculate how our observations may support the idea that the polymerisation process may be occurring at the ends of the chains with monomers adding sequentially. Our findings will contribute towards the development of new biomaterials for 3D cell culture and bio-printing.

Hydrogels of engineered bacterial fimbriae can finely tune 2D human cell culture.

Demand continues to grow for biomimetic materials able to create well-defined environments for modulating the behaviour of living cells in culture. Here, we describe hydrogels based upon the polymeric bacterial fimbriae protein capsular antigen fragment 1 (Caf1) that presents tunable biological properties for enhanced tissue cell culture applications. We demonstrate how Caf1 hydrogels can regulate cellular functions such as spreading, proliferation and matrix deposition of human dermal fibroblast cells (hDFBs). Caf1 hydrogels exploring a range of mechanical properties were prepared using copolymers featuring controlled compositions of inert wild-type Caf1 subunits and a mutant subunit displaying the RGDS peptide motif. The hydrogels showed excellent cytocompatibility with hDFBs and the ability to modulate both cell morphology and matrix deposition. Interestingly, Caf1 hydrogels displaying faster stress relaxation were demonstrated to show the highest metabolic activities of growing cells in comparison with other Caf1 hydrogel formulations. The stiffest Caf1 hydrogel impacted cellular morphology, inducing alignment of the cells. This work is significant as it clearly indicates that Caf1-based hydrogels offer tuneable biochemical and mechanical substrates conditions suitable for cell culture applications.

Rational Design and Self-Assembly of Coiled-Coil Linked SasG Protein Fibrils.

Protein engineering is an attractive approach for the self-assembly of nanometer-scale architectures for a range of potential nanotechnologies. Using the versatile chemistry provided by protein folding and assembly, coupled with amino acid side-chain functionality, allows for the construction of precise molecular "protein origami" hierarchical patterned structures for a range of nanoapplications such as stand-alone enzymatic pathways and molecular machines. The Staphyloccocus aureus surface protein SasG is a rigid, rod-like structure shown to have high mechanical strength due to "clamp-like" intradomain features and a stabilizing interface between the G5 and E domains, making it an excellent building block for molecular self-assembly. Here we characterize a new two subunit system composed of the SasG rod protein genetically conjugated with de novo designed coiled-coils, resulting in the self-assembly of fibrils. Circular dichroism (CD) and quartz-crystal microbalance with dissipation (QCM-D) are used to show the specific, alternating binding between the two subunits. Furthermore, we use atomic force microscopy (AFM) to study the extent of subunit polymerization in a liquid environment, demonstrating self-assembly culminating in the formation of linear macromolecular fibrils.

Engineered mosaic protein polymers; a simple route to multifunctional biomaterials.

BACKGROUND: Engineered living materials (ELMs) are an exciting new frontier, where living organisms create highly functional materials. In particular, protein ELMs have the advantage that their properties can be manipulated via simple molecular biology. Caf1 is a protein ELM that is especially attractive as a biomaterial on account of its unique combination of properties: bacterial cells export it as a massive, modular, non-covalent polymer which is resistant to thermal and chemical degradation and free from animal material. Moreover, it is biologically inert, allowing the bioactivity of each 15 kDa monomeric Caf1 subunit to be specifically engineered by mutagenesis and co-expressed in the same Escherichia coli cell to produce a mixture of bioactive Caf1 subunits. RESULTS: Here, we show by gel electrophoresis and transmission electron microscopy that the bacterial cells combine these subunits into true mosaic heteropolymers. By combining two separate bioactive motifs in a single mosaic polymer we demonstrate its utility by stimulating the early stages of bone formation by primary human bone marrow stromal cells. Finally, using a synthetic biology approach, we engineer a mosaic of three components, demonstrating that Caf1 complexity depends solely upon the variety of monomers available. CONCLUSIONS: These results demonstrate the utility of engineered Caf1 mosaic polymers as a simple route towards the production of multifunctional biomaterials that will be useful in biomedical applications such as 3D tissue culture and wound healing. Additionally, in situ Caf1 producing cells could create complex bacterial communities for biotechnology.

Induction of the immunoprotective coat of Yersinia pestis at body temperature is mediated by the Caf1R transcription factor.

BACKGROUND: Thermal regulation of gene expression occurs in many microorganisms, and is mediated via several typical mechanisms. Yersinia pestis is the causative agent of the plague and spreads by zoonotic transfer from fleas to mammalian blood with a concomitant rapid temperature change, from ambient to 37 °C, which induces the expression of capsular antigen (Caf1) that inhibits phagocytosis. Caf1 is formed into long polymeric fimbriae by a periplasmic chaperone (Caf1M) and outer membrane usher (Caf1A). All three are encoded on an operon regulated by an AraC-type transcription factor Caf1R. The aim of this study was to determine the role of Caf1R in the thermal control of caf1 operon gene expression. RESULTS: PCR analysis of cDNA demonstrated that the genes of the operon are transcribed as a single polycistronic mRNA. Bioinformatic analysis, supported by deletion mutagenesis, then revealed a region containing the promoter of this polycistronic transcript that was critical for Caf1 protein expression. Caf1R was found to be essential for Caf1 protein production. Finally, RT-PCR analysis and western blot experiments showed large, Caf1R dependent increases in caf1 operon transcripts upon a shift in temperature from 25 °C to 35 °C. CONCLUSIONS: The results show that thermal control of Caf1 polymer production is established at the transcriptional level, in a Caf1R dependent manner. This gives us new insights into how a virulent pathogen evades destruction by the immune system by detecting and responding to environmental changes.

Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism.

Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including energy metabolism, calcium homeostasis and apoptosis. Miro proteins (Miros) are "atypical" Ras superfamily GTPases that display unique domain architecture and subcellular localisation regulating mitochondrial transport, autophagy and calcium sensing. Here, we present systematic catalytic domain characterisation and structural analyses of human Miros. Despite lacking key conserved catalytic residues (equivalent to Ras Y32, T35, G60 and Q61), the Miro N-terminal GTPase domains display GTPase activity. Surprisingly, the C-terminal GTPase domains previously assumed to be "relic" domains were also active. Moreover, Miros show substrate promiscuity and function as NTPases. Molecular docking and structural analyses of Miros revealed unusual features in the Switch I and II regions, facilitating promiscuous substrate binding and suggesting the usage of a novel hydrolytic mechanism. The key substitution in position 13 in the Miros leads us to suggest the existence of an "internal arginine finger", allowing an unusual catalytic mechanism that does not require GAP protein. Together, the data presented here indicate novel catalytic functions of human Miro atypical GTPases through altered catalytic mechanisms.

Thermal stability and rheological properties of the 'non-stick' Caf1 biomaterial.

The ability to culture cells in three-dimensions has many applications, from drug discovery to wound healing. 3D cell culture methods often require appropriate scaffolds that mimic the cellular environments of different tissue types. The choice of material from which these scaffolds are made is of paramount importance, as its properties will define the manner in which cells interact with the scaffold. Caf1 is a protein polymer that is secreted from its host organism, Yersinia pestis, to enable escape from phagocytosis. In vitro, cells adhere poorly to the protein unless adhesion motifs are specifically introduced. Caf1 is a good candidate biomaterial due to its definable bioactivity, economical production and its ability to form hydrogels, through the use of cross-linkers. In this study, the thermostability of Caf1 was tested over a range of chemical conditions, and an initial characterisation of its rheological properties conducted in order to assess the suitability of Caf1 as a biomedical material. The results show that Caf1 retains its high thermostability even in harsh conditions such as extremes of pH, high salt concentrations and the presence of detergents. In solution, the concentrated polymer behaves as a complex viscous liquid. Due to these properties, Caf1 polymers are compatible with 3D bioprinting technologies and could be made to form a stimuli-responsive biomaterial that can alter its macrorheological properties in response to external factors. Caf1 biomaterials could therefore prove useful as 3D cell scaffolds for use in cell culture and wound repair.

Probing the Surfaces of Biomacromolecules with Polymer-Scaffolded Dynamic Combinatorial Libraries.

Methods to analyze and compare biomacromolecular surfaces are still in their relative infancy on account of the challenges involved in comparing surfaces computationally. We describe a systems chemistry approach that utilizes polymer-scaffolded dynamic combinatorial libraries to experimentally probe biomacromolecular surfaces in aqueous solution which provides feedback as to the nature of the surfaces, allowing the comparison of three globular proteins and a nucleic acid.

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases.

The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNA(Phe) and tRNA(Trp) show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids ("binding signatures") together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal "recognition" domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.