RESUMO
Brahma-related gene 1 (Brg1) is one of the two mutually exclusive catalytic subunits of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. Several roles of Brg1 have been described including acting as a tumor suppressor but also functioning in neural stem cell (NSC) maintenance, neural crest development, or differentiation of oligodendrocytes and Schwann cells. Here, we generated human glial fibrillary acidic protein (hGFAP)-cre::Brg1fl/fl mice to analyze the function of Brg1 in multipotential NSCs during late stages of neural development. hGFAP-cre::Brg1fl/fl mice died approximately 2 weeks after birth. Macroscopic examination revealed a severe hydrocephalus and a decreased brain weight caused by the loss of Brg1. The cerebellum of hGFAP-cre::Brg1fl/fl mice displayed disorganized cortical layers as well as a massive hypoplasia due to a dramatically reduced number of granule neurons. The cerebrum presented with less proliferative and more apoptotic precursor cells in the subventricular zone (SVZ). Furthermore, the cerebral cortex stood out with significantly thinned upper layers and with impressive dendrite pathology. Finally, the hippocampus was severely underdeveloped with only a sparse number of detectable neurons. We conclude that NSCs depend on Brg1 to give rise to major essential brain structures including the cerebellum, the cerebral cortex, and the hippocampus.
Assuntos
Cerebelo/crescimento & desenvolvimento , Córtex Cerebral/crescimento & desenvolvimento , DNA Helicases/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Cerebelo/patologia , Córtex Cerebral/patologia , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Humanos , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/patologia , Masculino , Camundongos Transgênicos , Células-Tronco Neurais/patologia , Neurônios/patologia , Neurônios/fisiologiaRESUMO
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.
Assuntos
Hiperventilação , Deficiência Intelectual , Fator de Transcrição 4 , Animais , Fácies , Hipocampo , Humanos , Deficiência Intelectual/genética , Camundongos , Neurônios , Fator de Transcrição 4/genéticaRESUMO
The TCF4 gene encodes for the basic helix-loop-helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.
Assuntos
Proteínas Hedgehog/genética , Meduloblastoma/genética , Mutação , Fator de Transcrição 4/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Fácies , Proteínas Hedgehog/metabolismo , Humanos , Hiperventilação/genética , Hiperventilação/metabolismo , Hiperventilação/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos Knockout , Fator de Transcrição 4/metabolismoRESUMO
Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB.
Assuntos
Metilação de DNA , Neuroblastoma/classificação , Neuroblastoma/genética , Transtornos do Olfato/classificação , Transtornos do Olfato/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Criança , Diagnóstico Diferencial , Feminino , Humanos , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Mutação , Transcriptoma , Adulto JovemRESUMO
In the original publication, the second name of the twentieth author was incorrect. It should read as 'Miguel Sáinz-Jaspeado'. The original publication of the article has been updated to reflect the change. This correction was authored by Ulrich Schüller on behalf of all authors of the original publication.
RESUMO
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 O(6)-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.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , PanitumumabeRESUMO
Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.
Assuntos
Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Leucemia Mieloide/imunologia , Receptores Fc/imunologia , ADP Ribose Transferases/imunologia , Idoso , Apoptose/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Feminino , Células HL-60 , Humanos , Imunoterapia/métodos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/imunologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/imunologia , Células U937 , Fatores de Virulência/imunologia , Exotoxina A de Pseudomonas aeruginosaAssuntos
Antígenos CD/imunologia , Imunoterapia/métodos , Imunotoxinas/uso terapêutico , Leucemia Mieloide/terapia , Receptores Fc/imunologia , Apoptose/efeitos dos fármacos , Humanos , Imunotoxinas/farmacologia , Leucemia Mieloide/patologia , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Células Tumorais CultivadasRESUMO
The zinc-finger transcription factor GLI2 is frequently amplified in childhood medulloblastoma of the Sonic-hedgehog type (SHH-MB), with or without amplification of NMYC or deletion of TP53. Despite the aggressive tumor behavior, tumorigenesis is not well understood, and adequate mouse models are lacking. Therefore, we generated mice with a GLI2 overexpression under control of the hGFAP-promoter. These mice died within 150 days. The majority only survived until postnatal day 40. They displayed severe cerebellar hypoplasia, cortical malformations, but no brain tumors, except for one out of 23 animals with an undifferentiated hindbrain lesion. Additional loss of p53 did not result in cerebellar tumors, but partially rescued the cerebellar phenotype induced by GLI2 overexpression. Similarly, the combination of GLI2 and NMYC was neither sufficient for the development of SHH-MB. We therefore assume that the development of childhood SHH-MB in mice is either occurring in cellular origins outside the hGFAP-positive lineage or needs additional genetic drivers.
RESUMO
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.
Assuntos
Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central , Glioma , Neoplasias Encefálicas/genética , Neoplasias do Sistema Nervoso Central/genética , Metilação de DNA/genética , Glioma/genética , Humanos , Linfócitos do Interstício Tumoral , Microambiente Tumoral/genéticaRESUMO
PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression. METHODS: Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 (6)-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. RESULTS: The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 +/- 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 +/- 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 +/- 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection. CONCLUSION: The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.
Assuntos
Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Imagem Molecular/métodos , Fenômenos Ópticos , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Alquil e Aril Transferases/genética , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Corantes/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Transporte Proteico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de TempoRESUMO
Medulloblastoma (MB) is the most frequent malignant brain tumour in children with a poor outcome. Divided into four molecular subgroups, MB of the Sonic hedgehog (SHH) subgroup accounts for approximately 25% of the cases and is driven by mutations within components of the SHH pathway, such as its receptors PTCH1 or SMO. A fraction of these cases additionally harbour PIK3CA mutations, the relevance of which is so far unknown. To unravel the role of Pik3ca mutations alone or in combination with a constitutively activated SHH signalling pathway, transgenic mice were used. These mice show mutated variants within Smo, Ptch1 or Pik3ca genes in cerebellar granule neuron precursors, which represent the cellular origin of SHH MB. Our results show that Pik3ca mutations alone are insufficient to cause developmental alterations or to initiate MB. However, they significantly accelerate the growth of Shh MB, induce tumour spread throughout the cerebrospinal fluid, and result in lower survival rates of mice with a double Pik3caH1047R/SmoM2 or Pik3caH1047R/Ptch1 mutation. Therefore, PIK3CA mutations in SHH MB may represent a therapeutic target for first and second line combination treatments.
Assuntos
Neoplasias Cerebelares/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Meduloblastoma/genética , Mutação , Animais , Neoplasias Cerebelares/patologia , Modelos Animais de Doenças , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/patologia , Camundongos Transgênicos , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/genética , Neoplasias Experimentais/mortalidade , Receptor Patched-1/genética , Receptor Smoothened/genética , Neoplasias da Medula Espinal/secundário , Taxa de Sobrevida , Sequenciamento Completo do GenomaRESUMO
In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.
Assuntos
Apoptose/fisiologia , Corantes Fluorescentes/química , Coloração e Rotulagem/métodos , Marcadores de Afinidade/química , Anexina A5/química , Anexina A5/metabolismo , Células Sanguíneas/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Humanos , Neoplasias/metabolismo , Proteínas/química , Proteínas/metabolismo , TecnologiaRESUMO
CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within the CBP gene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBPFl/Fl, mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients' quality of life.
Assuntos
Proteína de Ligação a CREB/deficiência , Movimento Celular/fisiologia , Células-Tronco Neurais/metabolismo , Síndrome de Rubinstein-Taybi/metabolismo , Ativação Transcricional/fisiologia , Animais , Proteína de Ligação a CREB/genética , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Estudos Retrospectivos , Síndrome de Rubinstein-Taybi/diagnóstico por imagem , Síndrome de Rubinstein-Taybi/genéticaRESUMO
PURPOSE: Treatment of cancer using standard chemotherapy still offers a poor prognosis combined with severe side effects. Novel antibody-based therapies have been shown to overcome low efficiency and lack of selectivity by targeting cancer-associated antigens, such as aminopeptidase CD13. METHODS: We isolated a high-affinity CD13-specific single-chain fragment variable (scFv13) from a phage display library of V-genes from mice immunized with soluble antigen. An immunotoxin comprising the scFv13 and a truncated version of the exotoxin A of Pseudomonas aeruginosa (ETA', scFv13-ETA') and a bispecific scFv targeting CD13 and CD16 simultaneously (bsscFv[13xds16]) was generated and investigated for their therapeutic potential. RESULTS: Both fusion proteins bound specifically to target cells with high affinity. Furthermore, scFv13-ETA' inhibited the proliferation of human cancer cell lines efficiently at low concentrations (IC50 values of 408 pM-7 nM) and induced apoptosis (40-85% of target cells). The bsscFv triggered dose-dependent antibody-dependent cell-mediated cytotoxicity, resulting in the lysis of up to 23.9% A2058 cells, 18.0% MDA-MB-468 cells and 19.1% HL-60 cells. CONCLUSION: The provided data demonstrate potent therapeutic activity of the scFv13-ETA' and the bsscFv[13xds16]. The CD13-specific scFv is therefore suitable for the direct and specific delivery of both cytotoxic agents and effector cells to cancer-derived cells, making it ideal for further therapeutic evaluation.
Assuntos
ADP Ribose Transferases/imunologia , Anticorpos Biespecíficos/farmacologia , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/imunologia , Antígenos CD13/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Exotoxinas/imunologia , Imunotoxinas/farmacologia , Neoplasias/tratamento farmacológico , Anticorpos de Cadeia Única/imunologia , Fatores de Virulência/imunologia , Antígenos CD13/imunologia , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Recombinantes/farmacologia , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
Antibody-drug conjugates (ADCs) can deliver toxins to specific targets such as tumor cells. They have shown promise in preclinical/clinical development but feature stoichiometrically undefined chemical linkages, and those based on full-size antibodies achieve only limited tumor penetration. SNAP-tag technology can overcome these challenges by conjugating benzylguanine-modified toxins to single-chain fragment variables (scFvs) with 1:1 stoichiometry while preserving antigen binding. Two (human and mouse) scFv-SNAP fusion proteins recognizing the epidermal growth factor receptor (EGFR) were expressed in HEK 293T cells. The purified fusion proteins were conjugated to auristatin F (AURIF). Binding activity was confirmed by flow cytometry/immunohistochemistry, and cytotoxic activity was confirmed by cell viability/apoptosis and cell cycle arrest assays, and a novel microtubule dynamics disassembly assay was performed. Both ADCs bound specifically to their target cells in vitro and ex vivo, indicating that the binding activity of the scFv-SNAP fusions was unaffected by conjugation to AURIF. Cytotoxic assays confirmed that the ADCs induced apoptosis and cell cycle arrest at nanomolar concentrations and microtubule disassembly. The SNAP-tag technology provides a platform for the development of novel ADCs with defined conjugation sites and stoichiometry. We achieved the stable and efficient linkage of AURIF to human or murine scFvs using the SNAP-tag technology, offering a strategy to improve the development of personalized medicines.
RESUMO
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™ 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.
Assuntos
Linfócitos B/citologia , Engenharia Celular/métodos , Granzimas/genética , Toxoide Tetânico/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Granzimas/metabolismo , Humanos , Imunotoxinas/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Toxoide Tetânico/metabolismoRESUMO
Human cytolytic fusion proteins (hCFPs) offer a promising immunotherapeutic approach for the treatment of solid tumors, avoiding the immunogenicity and undesirable side-effects caused by immunotoxins derived from plants or bacteria. The well-characterized human serine protease granzyme B has already been used as a therapeutic pro-apoptotic effector domain. We therefore developed a novel recombinant hCFP (GbR201K-scFv1711) consisting of an epidermal growth factor receptor-specific human antibody fragment and a granzyme B point mutant (R201K) that is insensitive to serpin B9 (PI9), a natural inhibitor of wild-type granzyme B that is often expressed in solid tumors. We found that GbR201K-scFv1711 selectively bound to epidermoid cancer and rhabdomyosarcoma cells and was rapidly internalized by them. Nanomolar concentrations of GbR201K-scFv1711 achieved the specific killing of epidermoid cancer cells by inducing apoptosis, and similar effects were observed in rhabdomyosarcoma cells when GbR201K-scFv1711 was combined with the endosomolytic substance chloroquine. The novel hCFP was stable in serum and bound to human rhabdomyosarcoma tissue ex vivo. These data confirm that GbR201K-scFv1711 is a promising therapeutic candidate suitable for further clinical investigation.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Receptores ErbB/biossíntese , Granzimas/farmacologia , Imunotoxinas/farmacologia , Linfoma/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/farmacologia , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cloroquina/farmacologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Granzimas/genética , Células HEK293 , Humanos , Imunotoxinas/genética , Imunotoxinas/imunologia , Linfoma/enzimologia , Linfoma/imunologia , Linfoma/patologia , Masculino , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Células U937RESUMO
Antibody-drug conjugates (ADCs) combine the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). Most ADCs are currently generated by the nonspecific conjugation of drug-linker reagents to certain amino acid residues in mAbs, resulting in a heterogeneous product. To overcome this limitation and prepare ADCs with a defined stoichiometry, we use SNAP-tag technology as an alternative conjugation strategy. This allows the site-specific conjugation of O(6)-benzylguanine (BG)-modified small molecules to SNAP-tag fusion proteins. To demonstrate the suitability of this system for the preparation of novel recombinant ADCs, here we conjugated SNAP-tagged single chain antibody fragments (scFvs) to a BG-modified version of auristatin F (AURIF). We used two scFv-SNAP fusion proteins targeting members of the epidermal growth factor receptor (EGFR) family that are frequently overexpressed in breast cancer. The conjugation of BG-AURIF to EGFR-specific 425(scFv)-SNAP and HER2-specific αHER2(scFv)-SNAP resulted in two potent recombinant ADCs that specifically killed breast cancer cell lines by inducing apoptosis when applied at nanomolar concentrations. These data confirm that SNAP-tag technology is a promising tool for the generation of novel recombinant ADCs.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptores ErbB/imunologia , Guanina/análogos & derivados , Imunoterapia/métodos , Imunotoxinas/farmacologia , Oligopeptídeos/farmacologia , Proteínas Q-SNARE/farmacologia , Receptor ErbB-2/imunologia , Anticorpos de Cadeia Única/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Receptores ErbB/metabolismo , Guanina/farmacologia , Humanos , Imunotoxinas/imunologia , Concentração Inibidora 50 , Camundongos , Proteínas Q-SNARE/imunologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Anticorpos de Cadeia Única/imunologiaRESUMO
PURPOSE: Rhabdomyosarcoma (RMS) is a rare and aggressive soft tissue sarcoma with limited treatment options and a high failure rate during standard therapy. New therapeutic strategies based on targeted immunotherapy are therefore much in demand. The epidermal growth factor receptor (EGFR) has all the characteristics of an ideal target. It is overexpressed in up to 80 % of embryonal RMS and up to 50 % of alveolar RMS tumors. We therefore tested the activity of the EGFR-specific recombinant immunotoxin (IT) 425(scFv)-ETA' against EGFR(+) RMS cells in vitro and ex vivo. METHODS: We tested the specific binding and internalization behavior of 425(scFv)-ETA' in RMS cell lines in vitro by flow cytometry, compared to the corresponding imaging probe 425(scFv)-SNAP monitored by live cell imaging. The cytotoxic activity of 425(scFv)-ETA' was tested using cell viability and apoptosis assays. Specific binding of the IT was confirmed on formalin-fixed paraffin-embedded tissue samples from two RMS patients. RESULTS: We confirmed the specific binding of 425(scFv)-ETA' to RMS cells in vitro and ex vivo. Both the IT and the corresponding imaging probe were rapidly internalized. The IT killed EGFR(+) RMS cells in a dose-dependent manner, while showing no effect against control cells. It showed specific apoptotic activity against one selected RMS cell line. CONCLUSIONS: This is the first study showing the promising therapeutic potential of a recombinant, EGFR-targeting, ETA'-based IT on RMS cells. We confirmed the selective killing with IC50 values of up to 50 pM, and immunohistochemical staining confirmed the specific ex vivo binding to primary RMS material.