CAR T cells for treating autoimmune diseases.

Autoimmune disorders occur when immune cells go wrong and attack the body's own tissues. Currently, autoimmune disorders are largely treated by broad immunosuppressive agents and blocking antibodies, which can manage the diseases but often are not curative. Thus, there is an urgent need for advanced therapies for patients suffering from severe and refractory autoimmune diseases, and researchers have considered cell therapy as potentially curative approach for several decades. In the wake of its success in cancer therapy, adoptive transfer of engineered T cells modified with chimeric antigen receptors (CAR) for target recognition could now become a therapeutic option for some autoimmune diseases. Here, we review the ongoing developments with CAR T cells in the field of autoimmune disorders. We will cover first clinical results of applying anti-CD19 and anti-B cell maturation antigen CAR T cells for B cell elimination in systemic lupus erythematosus, refractory antisynthetase syndrome and myasthenia gravis, respectively. Furthermore, in preclinical models, researchers have also developed chimeric autoantibody receptor T cells that can eliminate individual B cell clones producing specific autoantibodies, and regulatory CAR T cells that do not eliminate autoreactive immune cells but dampen their wrong activation. Finally, we will address safety and manufacturing aspects for CAR T cells and discuss mRNA technologies and automation concepts for ensuring the future availability of safe and efficient CAR T cell products.

Identification and characterization of novel CD274 (PD-L1) regulating microRNAs and their functional relevance in melanoma.

BACKGROUND: Immune checkpoint inhibitors directed against programmed cell death 1 (PDCD1/PD1) receptor and programmed cell death-ligand 1 (CD274/PD-L1) have been recently successfully implemented for the treatment of many cancers, but the response rate of tumour patients is still limited due to intrinsic and acquired resistances. However, the underlying molecular mechanisms of this limited response have still to be defined in detail. The aim of this study is to uncover processes inhibiting PDCD1/CD274 expression thereby enhancing anti-tumour immune responses. The identification and characterization of microRNAs (miRNAs) targeting the 3'-untranslated region (3'-UTR) as well as the coding sequence (CDS) of CD274 will provide the basis for a new drug development. METHODS: Human melanoma cell lines and tissue samples were subjected to mRNA and/or protein expression analysis using qPCR, Western blot, flow cytometry, and/or immunohistochemistry. The data were correlated to clinical parameters. MiRNA trapping by RNA in vitro affinity purification (miTRAP) technology in combination with small RNA sequencing and different bioinformatics tools were employed to identify CD274-regulating miRNAs. RESULTS: Screening based on miTRAP in combination with RNAseq identified a large number of novel CD274-regulating candidate miRNAs, from which eight selected miRNAs were functionally validated. Five out of eight miRNAs were able to significantly reduce CD274 surface expression indicating that these miRNAs directly bind to the 3'-UTR or CDS of the CD274 gene. The miRNA-mediated inhibition of CD274 expression was accompanied by an increased T cell recognition. Furthermore, an inverse expression of three CD274-regulating miRNAs and CD274 was demonstrated in melanoma lesions. A CD274 miRNA score was generated, which was associated with disease progression and reduced survival of melanoma patients. CONCLUSIONS: These data revealed a novel mechanism that miRNAs targeting the CDS of immune checkpoint genes are functional, have prognostic relevance, and also the potential for the development of novel miRNA-based therapies.

Identification of microRNAs Targeting the Transporter Associated with Antigen Processing TAP1 in Melanoma.

The underlying molecular mechanisms of the aberrant expression of components of the HLA class I antigen processing and presentation machinery (APM) in tumors leading to evasion from T cell-mediated immune surveillance could be due to posttranscriptional regulation mediated by microRNAs (miRs). So far, some miRs controlling the expression of different APM components have been identified. Using in silico analysis and an miR enrichment protocol in combination with small RNA sequencing, miR-26b-5p and miR-21-3p were postulated to target the 3' untranslated region (UTR) of the peptide transporter TAP1, which was confirmed by high free binding energy and dual luciferase reporter assays. Overexpression of miR-26b-5p and miR-21-3p in melanoma cells downregulated the TAP1 protein and reduced expression of HLA class I cell surface antigens, which could be reverted by miR inhibitors. Moreover, miR-26b-5p overexpression induced a decreased T cell recognition. Furthermore, an inverse expression of miR-26b-5p and miR-21-3p with TAP1 was found in primary melanoma lesions, which was linked with the frequency of CD8+ T cell infiltration. Thus, miR-26-5p and miR-21-3p are involved in the HLA class I-mediated immune escape and might be used as biomarkers or therapeutic targets for HLA class Ilow melanoma cells.

Tumor-induced escape mechanisms and their association with resistance to checkpoint inhibitor therapy.

Immunotherapy aims to activate the immune system to fight cancer in a very specific and targeted manner. Despite the success of different immunotherapeutic strategies, in particular antibodies directed against checkpoints as well as adoptive T-cell therapy, the response of patients is limited in different types of cancers. This attributes to escape of the tumor from immune surveillance and development of acquired resistances during therapy. In this review, the different evasion and resistance mechanisms that limit the efficacy of immunotherapies targeting tumor-associated antigens presented by major histocompatibility complex molecules on the surface of the malignant cells are summarized. Overcoming these escape mechanisms is a great challenge, but might lead to a better clinical outcome of patients and is therefore currently a major focus of research.

TGF-ß inducible epithelial-to-mesenchymal transition in renal cell carcinoma.

Epithelial-to-mesenchymal transition (EMT) is a crucial step in cancer progression and the number one reason for poor prognosis and worse overall survival of patients. Although this essential process has been widely studied in many solid tumors as e.g. melanoma and breast cancer, more detailed research in renal cell carcinoma (RCC) is required, especially for the major EMT-inducer transforming growth factor beta (TGF-ß). Here, we provide a study of six different RCC cell lines of two different RCC subtypes and their response to recombinant TGF-ß1 treatment. We established a model system shifting the cells to a mesenchymal cell type without losing their mesenchymal character even in the absence of the external stimulus. This model system forms a solid basis for future studies of the EMT process in RCCs to better understand the molecular basis of this process responsible for cancer progression.

The ancestor of modern Holozoa acquired the CCA-adding enzyme from Alphaproteobacteria by horizontal gene transfer.

Transfer RNAs (tRNAs) require the absolutely conserved sequence motif CCA at their 3'-ends, representing the site of aminoacylation. In the majority of organisms, this trinucleotide sequence is not encoded in the genome and thus has to be added post-transcriptionally by the CCA-adding enzyme, a specialized nucleotidyltransferase. In eukaryotic genomes this ubiquitous and highly conserved enzyme family is usually represented by a single gene copy. Analysis of published sequence data allows us to pin down the unusual evolution of eukaryotic CCA-adding enzymes. We show that the CCA-adding enzymes of animals originated from a horizontal gene transfer event in the stem lineage of Holozoa, i.e. Metazoa (animals) and their unicellular relatives, the Choanozoa. The tRNA nucleotidyltransferase, acquired from an α-proteobacterium, replaced the ancestral enzyme in Metazoa. However, in Choanoflagellata, the group of Choanozoa that is closest to Metazoa, both the ancestral and the horizontally transferred CCA-adding enzymes have survived. Furthermore, our data refute a mitochondrial origin of the animal tRNA nucleotidyltransferases.

The TRAMP complex shows tRNA editing activity in S. cerevisiae.

Transfer RNA (tRNA) editing is a widespread processing phenomenon that alters the sequence of primary transcripts by base substitutions as well as nucleotide deletions and insertions at internal or terminal transcript positions. In the corresponding tRNAs, these events are an important prerequisite for the generation of functional transcripts. Although many editing events are well characterized at the reaction level, it is unclear in most cases from which ancestral activities the modern editing enzymes evolved. Here, we show that in Saccharomyces cerevisiae, the noncanonical poly(A) polymerase Trf4p in the TRAMP complex can be recruited for such an editing reaction at an introduced tRNA transcript. As a distributive polymerase involved in RNA surveillance and quality control, it has a broad substrate spectrum and binds only transiently to the transcripts, limiting the number of added nucleotides at the editing position. These features exactly meet the criteria for an ancestral enzyme of a modern editing activity. Accordingly, our observations are a strong experimental support for the hypothesis that enzymatic promiscuity serves as an evolutionary starting point for the emergence of new functions and activities.

An inhibitory C-terminal region dictates the specificity of A-adding enzymes.

For efficient aminoacylation, tRNAs carry the conserved 3'-terminal sequence C-C-A, which is synthesized by highly specific tRNA nucleotidyltransferases (CCA-adding enzymes). In several prokaryotes, this function is accomplished by separate enzymes for CC- and A-addition. As A-adding enzymes carry an N-terminal catalytic core identical to that of CCA-adding enzymes, it is unclear why their activity is restricted. Here, it is shown that C-terminal deletion variants of A-adding enzymes acquire full and precise CCA-incorporating activity. The deleted region seems to be responsible for tRNA primer selection, restricting the enzyme's specificity to tRNAs ending with CC. The data suggest that A-adding enzymes carry an intrinsic CCA-adding activity that can be reactivated by the introduction of deletions in the C-terminal domain. Furthermore, a unique subtype of CCA-adding enzymes could be identified that evolved out of A-adding enzymes, suggesting that mutations and deletions in nucleotidyltransferases can lead to altered and even more complex activities, as a simple A-incorporation is converted into sequence-specific addition of C and A residues. Such activity-modifying events may have had an important role in the evolution of tRNA nucleotidyltransferases.