Retargeting of UniCAR T cells with an in vivo synthesized target module directed against CD19 positive tumor cells

Recent treatments of leukemias with T cells expressing chimeric antigen receptors (CARs) underline their impressive therapeutic potential but also their risk of severe side effects including cytokine release storms and tumor lysis syndrome. In case of cross-reactivities, CAR T cells may also attack healthy tissues. To overcome these limitations, we previously established a switchable CAR platform technology termed UniCAR. UniCARs are not directed against typical tumor-associated antigens (TAAs) but instead against a unique peptide epitope: Fusion of this peptide epitope to a recombinant antibody domain results in a target module (TM). TMs can cross-link UniCAR T cells with tumor cells and thereby lead to their destruction. So far, we constructed TMs with a short half-life. The fast turnover of such a TM allows to rapidly interrupt the treatment in case severe side effects occur. After elimination of most of the tumor cells, however, longer lasting TMs which have not to be applied via continous infusion would be more convenient for the patient. Here we describe and characterize a TM for retargeting UniCAR T cells to CD19 positive tumor cells. Moreover, we show that the TM can efficiently be produced in vivo from producer cells housed in a sponge-like biomimetic cryogel and, thereby, serving as an in vivo TM factory for an extended retargeting of UniCAR T cells to CD19 positive leukemic cells

Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

Analysis of the transcription factor TGA2.1 from Nicotiana tabacum

Zur Aufklärung der spezifischen Rolle von TGA2.1 sollten transgene TGA2.1-Überexpressionspflanzen sowie TGA2.1-transdominante Suppressor-Linien erzeugt werden. Die subzellullare Lokalisation der transgenen Proteine sollte durch Western-Blot-Analysen und DNA-Bindungsstudien untersucht werden. Weiterhin sollte die Auswirkung der veränderten TGA2.1-Mengen auf Zielgene nach Induktion mit Salizylsäure, Auxin und Methyljasmonat untersucht und die Ergebnisse mit denen analoger Experimente mit TGA2.2-Überexpressionspflanzen verglichen werden. Komplementierend zu den Überexpressionspflanzen sollten transdominante Suppressorpflanzen mit einem in der DNA-Bindungsdomäne mutierten TGA2.1 erzeugt werden. Die analogen Experimente wie im Falle der TGA2.1-Überexpressionpflanzen sollten das Bild von der spezifischen Rolle von TGA2.1 ergänzen und im Zusammenhang mit den Ergebnissen aus bereits vorliegenden TGA2.2-Experimenten interpretiert werden. Da in früheren Veröffentlichungen nicht eindeutig nachzugewiesen werden konnte, ob der SARP-Komplex neben TGA2.2 auch TGA2.1 enthält oder positive Nachweisreaktionen mit einer Kreuzreaktionen des Antikörpers zu erklären waren, und ob die im Western-Blot detektierte Bande somit als gekürzte Formen von TGA2.1 oder durch ein weiteres Protein zu interpretieren war, sollte die Zusammensetzung des SARP-Komplexes untersucht werden. Sollte sich herausstellen, dass es sich bei den nachgewiesenen Protein um TGA2.1 handelt, galt es zu klären, ob die Prozessierung des Proteins eine physiologische Relevanz hat oder ein Produkt des Zellaufschlusses ist. Im letzteren Fall sollten geeignetere Proteinextraktionsmethoden entwickelt werden. Ein Affinitätstag am C-Terminus von TGA2.1 sollte verwendet werden, um die absoluten Mengen des transgenen TGA2.1 mit denen des transgenen TGA2.2 zu vergleichen und um darüber hinaus die Lokalisation der möglichen Deletion von TGA2.1 zu klären. Da bei den vorliegenden transgenen TGA2.2-Pflanzen die Zellkernproteinfraktion nicht untersucht worden war, wurden diese in die Untersuchungen des ASF-1-Komplexes mit einbezogen

Artificial splitting of a non‐ribosomal peptide synthetase by inserting natural docking domains

The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme

Insect-specific production of new gameXpeptides in photorhabdus luminescensTTO1, widespread natural products in entomopathogenic bacteria

Discovery of new natural products by heterologous expression reaches its limits, especially when specific building blocks are missing in the heterologous host or the production medium. Here, we describe the insect-specific production of the new GameXPeptides E–H (5–8) from Photorhabdus luminescens TTO1, which can be produced heterologously from expression of the GameXPeptide synthetase GxpS only upon supplementation of the production media with the missing building blocks, and thus must be regarded as the true natural products under natural conditions

Structure-based redesign of docking domain interactions modulates the product spectrum of a rhabdopeptide-synthesizing NRPS

Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs ((N)DDs) as well as of an (DD)-D-N-(DD)-D-C complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes

Radical S-Adenosyl Methionine Epimerases: Regioselective Introduction of Diverse D -Amino Acid Patterns into Peptide Natural Products

International audiencePoyD is a radical S-adenosyl methionine epimerase that introduces multiple d-configured amino acids at alternating positions into the highly complex marine peptides poly-theonamide A and B. This novel post-translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse d-amino acid patterns into all-l peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering