Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding ...of a small five-helix protein domain-the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK-in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.
tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient ...starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic Formula (tK ᵁᵁᵁ), Formula (tQ ᵁᵁᴳ), and Formula (tE ᵁᵁC). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1 - and ELP -dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK ᵁᵁᵁ to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.
Speed and accuracy of protein synthesis are fundamental parameters for the fitness of living cells, the quality control of translation, and the evolution of ribosomes. The ribosome developed complex ...mechanisms that allow for a uniform recognition and selection of any cognate aminoacyl-tRNA (aa-tRNA) and discrimination against any near-cognate aa-tRNA, regardless of the nature or position of the mismatch. This review describes the principles of the selection—kinetic partitioning and induced fit—and discusses the relationship between speed and accuracy of decoding, with a focus on bacterial translation. The translational machinery apparently has evolved towards high speed of translation at the cost of fidelity.
Improving the yield of unnatural amino acid incorporation is an important challenge in producing novel designer proteins with unique chemical properties. Here we examine the mechanisms that restrict ...the incorporation of the fluorescent unnatural amino acid εNH2-Bodipy576/589-lysine (BOP-Lys) into a model protein. While the delivery of BOP-Lys-tRNALys to the ribosome is limited by its poor binding to elongation factor Tu (EF-Tu), the yield of incorporation into peptide is additionally controlled at the step of BOP-Lys-tRNA release from EF-Tu into the ribosome. The unnatural amino acid appears to disrupt the interactions that balance the strength of tRNA binding to EF-Tu-GTP with the velocity of tRNA dissociation from EF-Tu-GDP on the ribosome, which ensure uniform incorporation of standard amino acids. Circumventing this potential quality control checkpoint that specifically prevents incorporation of unnatural amino acids into proteins may provide a new strategy to increase yields of unnatural polymers.
Selective gene delivery to a cell type of interest utilizing targeted lentiviral vectors (LVs) is an efficient and safe strategy for cell and gene therapy applications, including chimeric antigen ...receptor (CAR)-T cell therapy. LVs pseudotyped with measles virus envelope proteins (MV-LVs) have been retargeted by ablating binding to natural receptors while fusing to a single-chain antibody specific for the antigen of choice. However, the broad application of MV-LVs is hampered by the laborious LV engineering required for every new target. Here, we report the first versatile targeting system for MV-LVs that solely requires mixing with biotinylated adapter molecules to enable selective gene transfer. The analysis of the selectivity in mixed cell populations revealed transduction efficiencies below the detection limit in the absence of an adapter and up to 5000-fold on-to-off-target ratios. Flexibility was confirmed by transducing cell lines and primary cells applying seven different adapter specificities in total. Furthermore, adapter mixtures were applied to generate CAR-T cells with varying CD4/CD8-ratios in a single transduction step. In summary, a selective and flexible targeting system was established that may serve to improve the safety and efficacy of cellular therapies. Compatibility with a wide range of readily available biotinylated molecules provides an ideal technology for a variety of applications.
Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed.
Here, ...we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models.
AdCAR NK-92 cells may provide an interesting and promising "off-the-shelf" cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.
Despite the recent success of CAR T cells targeting CD19 and CD22 in hematological malignancies, the production of CAR T cells still requires an extensive manufacturing process. The well-established ...NK-92 cell line provides a promising alternative to produce CAR-modified effector cells in a GMP-compliant, cost-effective way. NK-92 can be redirected against a variety of surface antigens by our adapter CAR (AdCAR) system utilizing biotinylated antibodies (bAb) as adapter molecules. Selected bAb were capable of inducing significant AdCAR NK-92-mediated lysis of non-Hodgkin lymphoma (NHL) and mantle-cell lymphoma (MCL) cell lines as well as primary MCL and chronic lymphocytic leukemia (CLL) cells. AdCAR specificity was proven using a JeKo-1 CD19/CD20 knockout antigen-loss model. Moreover, through combinations of bAb, AdCAR NK-92 cells are capable of combatting tumor antigen evasion mechanisms. In conclusion, we successfully generated the AdCAR NK-92 cell line which can be manufactured as an "off-the-shelf, on-demand" product allowing universal and tunable tumor targeting.
Chimeric antigen receptor (CAR)-T therapy holds great promise to sustainably improve cancer treatment. However, currently, a broad applicability of CAR-T cell therapies is hampered by limited CAR-T ...cell versatility and tractability and the lack of exclusive target antigens to discriminate cancerous from healthy tissues. To achieve temporal and qualitative control on CAR-T function, we engineered the Adapter CAR (AdCAR) system. AdCAR-T are redirected to surface antigens via biotin-labeled adapter molecules in the context of a specific linker structure, referred to as Linker-Label-Epitope. AdCAR-T execute highly specific and controllable effector function against a multiplicity of target antigens. In mice, AdCAR-T durably eliminate aggressive lymphoma. Importantly, AdCAR-T might prevent antigen evasion by combinatorial simultaneous or sequential targeting of multiple antigens and are capable to identify and differentially lyse cancer cells by integration of adapter molecule-mediated signals based on multiplex antigen expression profiles. In consequence the AdCAR technology enables controllable, flexible, combinatorial, and selective targeting.
Adapter CAR-T cells for multiple synchronic targeting.
Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor ...control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.
Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble ...factors such as TGF-β. TGF-β plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-β within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-β, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-β and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-β.
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