The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt–Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS

Zielgerichtete therapeutische und diagnostische Ansätze für solide Tumore am Beispiel des EGF-Rezeptors

Solid tumors are difficult to treat, especially when they reach a metastatic stage. Standard therapies often show severe side-effects. Therefore, the evaluation of new and efficient treatment options are in urgent need. With targeted therapies using immunotoxins (IT) or in best case human cytolytic fusion proteins (hCFP), tumor cells could be killed specifically without affecting healthy cells. Hence, this work was focused on the evaluation of a Granzyme B (GrB)-based hCFP, which could induce apoptotic signal-cascades in tumor cells. GrB was fused to a cell-binding antibody-fragment, a new human epidermal growth factor receptor (EGFR)-specific single-chain Fragment variable (scFv), to demonstrate binding and subsequent internalization by receptor bearing cells. The EGFR is expressed on a variety of solid tumors and is associated with poor prognosis. Rhabdomyosarcoma (RMS), pancreatic-, prostate-, and epoidermoid carcinoma, as well as the breast cancer type triple negative breast cancer (TNBC) are difficult to treat and were the main focus of this study. To initially analyze the functionality of the human and a second EGFR-specific scFv, both were fused to the SNAP-Tag and characterized. ScFv1711-SNAP was derived from Panitumumab and scFv2112-SNAP from Cetuximab, which are established EGFR-specific monoclonal antibodies (mAb) in the clinic. Both scFv-SNAP fusion proteins showed specific binding and internalization into the target cells within minutes. A critical factor concerning ITs and hCFPs is the affinity constant of the used scFvs. In order to use SPR for determining the affinity of the scFv-SNAP fusion proteins, a regenerative system was established. For this purpose, the fast and easy coupling reaction of BG-Biotin to the scFv-SNAP fusion was combined with the commercial available “Biotin CAPture kit”, which works on the basis of Streptavidin/Biotin binding. Both scFvs show KD-values of ~ 4 nM. Additionally the competitive behavior of the scFvs was tested and compared to the parental mAbs, respectively. To get a first hint of the in vivo-potential, scFv2112-SNAP was successfully tested as NIR-probe in a subcutaneous prostate cancer-mouse model. After the functional characterization of the new scFvs, both were fused to a truncated version of Pseudomonas Exotoxin A (ETA') as proof of concept. Using cell lines from different tumor entities and with varying EGFR-expression levels, specific binding, internalization within 30-60 min, as well as IC50-values in the picomolar range (4-460 pM depending on the cell line) were determined. The cell line with the highest EGFR-expression level was most efficiently eliminated in cell viability assays. Both ITs also induced statistically significant apoptosis. In addition, specific binding on human tumor biopsies could be confirmed. The aim of the study was to develop and evaluate an hCFP for the treatment of solid tumors. This was demonstrated by using GbR201K-scFv1711 with a Serpin B9-resistent mutated GrB-variant. After expression and purification with yields in the range of 17-30 mg/l protein, specific binding and internalization was confirmed on selected cell lines, by using enzymatically activated GbR201K-scFv1711. GbR201K-scFv1711 demonstrated a high serum stability, even after 72h. Significant apoptotic and anti-proliferative effects (IC50 = 133.3 nM) were reached with A431-cells (epidermoid carcinoma). By using the endosomolytic substance Chloroquine (CQ) induction of apoptosis and an IC50-value of 21 nM could be determined with RD-cells (RMS) as well. The IC50-value of A431-cells improved to 45 nM. Furthermore, specific binding on human RMS-biopsies was demonstrated. These data confirm that especially this novel hCFP is an interesting candidate for further pre-clinical studies

DIMEimmune: Robust estimation of infiltrating lymphocytes in CNS tumors from DNA methylation profiles

The interaction of CNS tumors with infiltrating lymphocytes plays an important role in their initiation and progression and might be related to therapeutic responses. Gene expression-based methods have been successfully used to characterize the tumor microenvironment. However, methylation data are now increasingly used for molecular diagnostics and there are currently only few methods to infer information about the microenvironment from this data type. Using an approach based on differential methylation and principal component analysis, we developed DIMEimmune (Differential Methylation Analysis for Immune Cell Estimation) to estimate CD4+ and CD8+ T cell abundance as well as tumor-infiltrating lymphocytes (TILs) scores from bulk methylation data. Well-established approaches based on gene expression data and immunohistochemistry-based lymphocyte counts were used as benchmarks. The comparison of DIMEimmune to the previously published MethylCIBERSORT and MeTIL algorithms showed an improved correlation with both gene expression-based and immunohistological results across different brain tumor types. Further, we applied our method to large datasets of glioma, medulloblastoma, atypical teratoid/rhabdoid tumors (ATRTs) and ependymoma. High-grade gliomas showed higher scores of tumor-infiltrating lymphocytes than lower-grade gliomas. There were overall only few tumor-infiltrating lymphocytes in medulloblastoma subgroups. ATRTs were highly infiltrated by lymphocytes, most prominently in the MYC subgroup. DIMEimmune-based estimates of TILs were a significant prognostic factor in the overall cohort of gliomas and medulloblastomas, but not within methylation-based diagnostic subgroups. To conclude, DIMEimmune allows for robust estimates of TIL abundance and might contribute to establishing them as a prognostic or predictive factor in future studies of CNS tumors

SNAP-Tag Technology : A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments

Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O6-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives.National Research Foundation (South Africa

Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

The antigen-specific targeting of autoreactive B cells via their unique B cell receptors (BCRs) is a novel and promising alternative to the systemic suppression of humoral immunity. We generated and characterized cytolytic fusion proteins based on an existing immunotoxin comprising tetanus toxoid fragment C (TTC) as the targeting component and the modified Pseudomonas aeruginosa exotoxin A (ETA') as the cytotoxic component. The immunotoxin was reconfigured to replace ETA' with either the granzyme B mutant R201K or MAPTau as human effector domains. The novel cytolytic fusion proteins were characterized with a recombinant human lymphocytic cell line developed using Transpo-mAb (TM) technology. Genes encoding a chimeric TTC-reactive immunoglobulin G were successfully integrated into the genome of the precursor B cell line REH so that the cells could present TTC-reactive BCRs on their surface. These cells were used to investigate the specific cytotoxicity of GrB(R201K)-TTC and TTC-MAPTau, revealing that the serpin proteinase inhibitor 9-resistant granzyme B R201K mutant induced apoptosis specifically in the lymphocytic cell line. Our data confirm that antigen-based fusion proteins containing granzyme B (R201K) are suitable candidates for the depletion of autoreactive B cells

Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing

This work was funded by the Biotechnology and Biological Sciences Research Council [grant numbers BB/G011400/1 and BB/K000314/1 (to M.F.W. and J.H.N.)], a Biotechnology and Biological Sciences Research Council-funded studentship to J.R. and a Medical Research Council-funded studentship to R.D.S.The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed 'CRISPR' (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unit-length crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes.Publisher PDFPeer reviewe