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Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.
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Movimento Celular , Glipicanas/química , Receptores de Netrina/química , Animais , Glipicanas/metabolismo , Humanos , Camundongos , Proteínas Mutantes , Receptores de Netrina/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos de Domínio Único , TrombospondinasRESUMO
The Simpson-Golabi-Behmel syndrome (SGBS; OMIM 312870) is an overgrowth/multiple congenital anomalies/dysplasia condition, inherited as an X-linked semi-dominant trait, with variable expressivity in males and reduced penetrance and expressivity in females. The clinical spectrum is broad, ranging from mild manifestations in both males and females to multiple malformations and neonatal death in the more severely affected cases. An increased risk of neoplasia is reported, requiring periodical surveillance. Intellectual development is normal in most cases. SGBS is caused by a loss-of-function mutation of the GPC3 gene, either deletions or point mutations, distributed all over the gene. Notably, GPC3 deletion/point mutations are not found in a significant proportion of clinically diagnosed SGBS cases. The protein product GPC3 is a glypican functioning as a receptor for Hh at the cell surface, involved in the Hh-Ptc-Smo signaling pathway, a regulator of cellular growth.
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Glypican-3 (GPC3) is markedly overexpressed in hepatocellular carcinoma (HCC) and not expressed in normal liver tissues. In this study, a novel peptide PET imaging agent ([18F]AlF-NOTA-IPB-GPC3P) was developed to target GPC3 expressed in tumors. The overall radiochemical yield of [18F]AlF-NOTA-IPB-GPC3P was 10-15 %, and its lipophilicity, expressed as the logD value at a pH of 7.4, was -1.18 ± 0.06 (n = 3). Compared to the previously reported tracer [18F]AlF-GP2633, [18F]AlF-NOTA-IPB-GPC3P exhibited higher cellular uptake (15.13 vs 5.96) and internalized rate (80.63 % vs 35.93 %) in Huh7 cells at 120 min. Micro-PET/CT and biodistribution studies further demonstrated that [18F]AlF-NOTA-IPB-GPC3P exhibited significantly increased tumor uptake and prolonged tumor residence in Huh7 tumors compared to [18F]AlF-GP2633 (4.66 ± 0.22 % ID/g vs 0.72 ± 0.09 % ID/g at 60 min, p < 0.001; 5.05 ± 0.23 % ID/g vs 0.35 ± 0.08 % ID/g at 120 min, p < 0.001, respectively). Furthermore, the tumor-to-organ ratios of [18F]AlF-NOTA-IPB-GPC3P surpassed those of [18F]AlF-GP2633. Our results support the utilization of [18F]AlF-NOTA-IPB-GPC3P as a PET imaging agent targeting the GPC3 receptor for tumor detection.
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Radioisótopos de Flúor , Glipicanas , Tomografia por Emissão de Pósitrons , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Radioisótopos de Flúor/química , Glipicanas/metabolismo , Compostos Heterocíclicos com 1 Anel , Neoplasias Hepáticas/diagnóstico por imagem , Camundongos Nus , Estrutura Molecular , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
Chimeric antigen receptor-expressing T (CAR-T) cells induce robust antitumor responses in patients with hematologic malignancies. However, CAR-T cells exhibit only limited efficacy against solid tumors such as hepatocellular carcinoma (HCC), partially due to their limited expansion and persistence. CD8+ T cells, as key components of the adaptive immune response, play a central role in antitumor immunity. Aerobic glycolysis is the main metabolic feature of activated CD8+ T cells. In the tumor microenvironment, however, the uptake of large amounts of glucose by tumor cells and other immunosuppressive cells can impair the activation of T cells. Only when tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment have a glycolytic advantage might the effector function of T cells be activated. Glucose transporter type 1 (GLUT1) and acylglycerol kinase (AGK) can boost glycolytic metabolism and activate the effector function of CD8+ T cells, respectively. In this study, we generated GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK for the treatment of HCC. GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK specifically and effectively lysed GPC3-positive tumor cells in vitro in an antigen-dependent manner. Furthermore, GLUT1 or AGK overexpression protected CAR-T cells from apoptosis during repeated exposures to tumor cells. Compared with second-generation CAR-T cells, GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK exhibited greater CD8+ T-cell persistence in vivo and better antitumor effects in HCC allograft mouse models. Finally, we revealed that GLUT1 or AGK maintained anti-apoptosis ability in CD8+ T cells via activation of the PI3K/Akt pathway. This finding might identify a therapeutic strategy for advanced HCC.
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Carcinoma Hepatocelular , Transportador de Glucose Tipo 1 , Glipicanas , Neoplasias Hepáticas , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Animais , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Transportador de Glucose Tipo 1/metabolismo , Humanos , Camundongos , Glipicanas/metabolismo , Glipicanas/imunologia , Imunoterapia Adotiva/métodos , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Microambiente Tumoral , ApoptoseRESUMO
Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.
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Comunicação Interventricular , Humanos , Aberrações Cromossômicas , Variações do Número de Cópias de DNA/genética , Predisposição Genética para Doença/genética , Comunicação Interventricular/genética , Mutação , Fatores de Transcrição/genéticaRESUMO
In recent years, the development of bispecific antibodies (bsAbs) has become a major trend in the biopharmaceutical industry. By simultaneously engaging two molecular targets, bsAbs have exhibited unique mechanisms of action that could lead to clinical benefits unattainable by conventional monoclonal antibodies. The type of structure used to construct a bsAb directly influences the distance, angle, degree of freedom, and affinity between the two antibody binding sites and the interaction between the two antigens or the cells where the antigens are located, which have been bound by the antibody. Consequently, the structure of the bsAb is one of the most vital factors affecting its function. Herein, we reported for the first time a novel basic module bsAb format, VFV (Variable domain-Fab-Variable domain). And then, the feasibility of the VFV format was demonstrated by constructing a series of engager-like basic module bsAbs. Next, a series of VFV bsAbs containing Fc (VFV-Ig), Fab (VFV-Fab), or Hinge (VFV-Hinge) were developed based on Hxb module, and all of them had adequate purity and activity. Finally, a T cell engager bsAb with the potential to overcome on-target off-tumor activity was constructed according to the structural characteristics of VFV, which validated that the VFV module can be used as a new brick for the construction of various bsAbs. In a word, the successful construction of this bsAb format for the first time not only enriches the arsenal of the bsAb format, but also provides inspiration for the construction of new bsAbs. Nevertheless, we are fully aware that as a proof-of-concept study, this paper has many shortcomings, and there is still a lot of work to be done to determine whether VFV can serve as a platform for drug development.
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Anticorpos Biespecíficos , Neoplasias , Humanos , Anticorpos Monoclonais , Sítios de Ligação de Anticorpos , Linfócitos TRESUMO
The placenta and tumors can exhibit a shared expression profile of proto-oncogenes. The basis of placenta-derived heat shock protein gp96, which induces prophylactic and therapeutic T cell responses against cancer including hepatocellular carcinoma (HCC), remains unknown. Here, we identified the associated long peptides from human placental gp96 using matrix-assisted laser desorption/ionization-time-of-flight and mass spectrometry and analyzed the achieved proteins through disease enrichment analysis. We found that placental gp96 binds to numerous peptides derived from 73 proteins that could be enriched in multiple cancer types. Epitope-harboring peptides from glypican 3 (GPC3) and paternally expressed gene 10 (PEG10) were the major antigens mediating anti-HCC T cell immunity. Molecular docking analysis showed that the GPC3- and PEG10-derived peptides, mainly obtained from the cytotrophoblast layer of the mature placenta, bind to the lumenal channel and client-bound domain of the gp96 dimer. Immunization with bone marrow-derived dendritic cells pulsed with recombinant gp96-GPC3 or recombinant gp96-PEG10 peptide complex induced specific T cell responses, and T cell transfusion led to pronounced growth inhibition of HCC tumors in nude mice. We demonstrated that the chaperone gp96 can capture antigenic peptides as an efficient approach for defining tumor rejection oncoantigens in the placenta and provide a basis for developing GPC3 and PEG10 peptide-based vaccines against HCC. This study provides insight into the underlying mechanism of the antitumor response mediated by embryonic antigens from fetal tissues, and this will incite more studies to identify potential tumor rejection antigens from placenta.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Feminino , Humanos , Camundongos , Gravidez , Antígenos de Neoplasias , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Hepatocelular/terapia , Proteínas de Ligação a DNA/metabolismo , Glipicanas , Neoplasias Hepáticas/terapia , Camundongos Nus , Simulação de Acoplamento Molecular , Peptídeos , Placenta/metabolismo , Proteínas de Ligação a RNARESUMO
Osteosarcoma (OS) is the most common bone malignancy without a reliable therapeutic target. Glypican-3 (GPC3) mutation and upregulation have been detected in multidrug resistant OS, and anti-GPC3 immunotherapy can effectively suppress the growth of organoids. Further profiling of GPC3 mutations and expression patterns in OS is of clinical significance. To address these issues, fresh OS specimens were collected from 24 patients for cancer-targeted next-generation sequencing (NGS) and three-dimensional patient-derived organoid (PDO) culture. A tumor microarray was prepared using 37 archived OS specimens. Immunohistochemical (IHC) staining was performed on OS specimens and microarrays to profile GPC3 and CD133 expression as well as intratumoral distribution patterns. RT-PCR was conducted to semiquantify GPC3 and CD133 expression levels in the OS tissues. Anti-GPC3 immunotherapy was performed on OS organoids with or without GPC3 expression and its efficacy was analyzed using multiple experimental approaches. No OS cases with GPC3 mutations were found, except for the positive control (OS-08). IHC staining revealed GPC3 expression in 73.77% (45/61) of OSs in weak (+; 29/45), moderate (++; 8/45), and strong (+++; 8/45) immunolabeling densities. The intratumoral distribution of GPC3-positive cells was variable in the focal (+; 10%-30%; 8/45), partial (++; 31%-70%; 22/45), and the most positive patterns (+++; >71%; 15/45), which coincided with CD133 immunolabeling (P = 9.89 × 10-10 ). The anti-GPC3 antibody efficiently inhibits Wnt/ß-catenin signaling and induces apoptosis in GPC3-positive PDOs and PDXs, as opposed to GPC3-negative PDOs and PDXs. The high frequency of GPC3 and CD133 co-expression and the effectiveness of anti-wild-type GPC3-Ab therapy in GPC3-positive OS models suggest that GPC3 is a novel prognostic parameter and a promising therapeutic target for osteosarcoma.
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Neoplasias Ósseas , Carcinoma Hepatocelular , Neoplasias Hepáticas , Osteossarcoma , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Carcinoma Hepatocelular/patologia , Glipicanas/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , beta CateninaRESUMO
CD3 bispecific constructs are anticipated to become an important form of cancer immunotherapy, but they frequently cause cytokine release syndrome (CRS) that is difficult to manage in clinical contexts. A combination of intra-patient dose escalation and immunosuppressive treatment is widely used to mitigate CRS. Studies suggest that CRS after subsequent doses of CD3 bispecific constructs is less severe than after the priming dose, and that step-up dosing reduces cytokine levels in animals and humans. However, the mechanism underlying the reduced cytokine induction after priming treatment with CD3 bispecific constructs is unclear. To understand human T-cell activation and chromatin states after priming treatment with CD3 bispecific construct targeting CD3É and glypican 3 (ERY974), we examined cytokine levels, cytokine mRNA expression, CD3É expression, CD3-mediated signal transduction, T cell activation markers, cytotoxicity against target cells, and chromatin states in T cells after ERY974 priming treatment or negative control. The second ERY974 treatment decreased cytokines on Day 8, and ERY974 priming treatment changed the chromatin state in T cells. CD3É expression, CD3-mediated signal transduction, T cell activation markers, and cytotoxicity were similar between the priming treatment with ERY974 and negative control. The present study suggests that chromatin state changes in T cells after the priming treatment was a pivotal factor in the mitigation of cytokine release after the second ERY974 treatment.
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Antineoplásicos , Linfócitos T , Animais , Anticorpos Biespecíficos , Antineoplásicos/farmacologia , Complexo CD3 , Cromatina , Síndrome da Liberação de Citocina , Citocinas/metabolismo , Humanos , Ativação LinfocitáriaRESUMO
PURPOSE: Early detection of hepatocellular carcinoma (HCC) remains a clinical challenge. Glypican 3 (GPC3) is a proteoglycan highly specific for HCC and is a potential diagnostic and therapeutic target for HCC. This work aims to develop GPC3-targeted immuno-positron emission tomography (immunoPET) imaging strategies and to assess the diagnostic values in preclinical HCC models. METHODS: Flow cytometry was used to screen GPC3-positive HCC cell lines. The expression of GPC3 in HCCs was detected by immunohistochemistry on tissue microarray. A novel GPC3-specific single domain antibody (sdAb) was produced and labeled with gallium-68 (68Ga, T1/2 = 1.1 h) and fluorine-18 (18F, T1/2 = 1.8 h) to develop radiotracers with different half-lives. The diagnostic efficacies of the developed probes (i.e., [68Ga]Ga-NOTA-G2, [18F]F-G2, and [68Ga]Ga-NOTA-ABDG2) were interrogated in preclinical HCC models bearing varying GPC3 levels. RESULTS: GPC3 was strongly expressed on HCC cell lines and patients with poorly differentiated HCC. [68Ga]Ga-NOTA-G2 immunoPET imaging specifically delineated the subcutaneous HCC lesions, outperforming the traditional 18F-fluorodeoxyglucose PET and the nonspecific [68Ga]Ga-NOTA-NbGFP immunoPET. ImmunoPET imaging with [18F]F-G2 also efficiently diagnosed the tumors with clarity. Moreover, the fusion of G2 to an albumin-binding domain (ABD) significantly increased the tumor uptake and decreased kidney accumulation of the radiotracer when compared to [68Ga]Ga-NOTA-G2. CONCLUSIONS: In the work, we successfully developed sdAb-derived GPC3-targeted immunoPET imaging strategies and characterized the superior diagnostic accuracies in preclinical HCC models. Furthermore, we synthesized a fusion protein ABDG2 with improved targeting and pharmacokinetic properties, serving as a promising candidate for developing radioimmunotherapy agents.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/metabolismo , Radioisótopos de Gálio , Glipicanas/química , Glipicanas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Tomografia por Emissão de PósitronsRESUMO
Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen, yet anti-GPC3 therapies have achieved only minimal clinical progress. CD47 is a ubiquitously expressed innate immune checkpoint that promotes evasion of tumors from immune surveillance. Given both the specific expression of GPC3 in HCC and the known phagocytosis inhibitory effect of CD47 in liver cancer, we hypothesized that a bispecific antibody (BsAb) that co-engages with GPC3 and CD47 may offer excellent antitumor efficacy with minimal toxicity. Here, we generated a novel BsAb: GPC3/CD47 biAb. With the use of both in vitro and in vivo assays, we found that GPC3/CD47 biAb exerts strong antitumor activity preferentially against dual antigen-expressing tumor cells. In hCD47/human signal regulatory protein alpha (hCD47/hSIRPα) humanized mice, GPC3/CD47 biAb had an extended serum half-life without causing systemic toxicity. Importantly, GPC3/CD47 biAb induced enhanced Fc-mediated effector functions to dual antigen-expressing HCC cells in vitro, and both macrophages and neutrophils are required for its strong efficacy against xenograft HCC tumors. Notably, GPC3/CD47 biAb outperformed monotherapies and a combination therapy with anti-CD47 and anti-GPC3 monoclonal antibodies (mAbs) in a xenograft HCC model. Our study illustrates a strategy for improving HCC treatment by boosting innate immune responses and presents new insights to inform antibody design for the future development of innovative immune therapies.
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Antígeno CD47/genética , Carcinoma Hepatocelular/tratamento farmacológico , Glipicanas/genética , Neoplasias Hepáticas/tratamento farmacológico , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/imunologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Glipicanas/antagonistas & inibidores , Glipicanas/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The pandemic of the novel coronavirus disease 2019 (COVID-19) is continuously causing hazards for the world. Effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can relieve the impact, but various toxic chemicals are also released into the environment. Fluorescence sensors offer a facile analytical strategy. During fluorescence sensing, biological samples such as tissues and body fluids have autofluorescence, giving false-positive/negative results because of the interferences. Fluorescence near-infrared (NIR) nanosensors can be designed from low-toxic materials with insignificant background signals. Although this research is still in its infancy, further developments in this field have the potential for sustainable detection of SARS-CoV-2. Herein, we summarize the reported NIR fluorescent nanosensors with the potential to detect SARS-CoV-2. The green synthesis of NIR fluorescent nanomaterials, environmentally compatible sensing strategies, and possible methods to reduce the testing frequencies are discussed. Further optimization strategies for developing NIR fluorescent nanosensors to facilitate greener diagnostics of SARS-CoV-2 for pandemic control are proposed.
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The acceleration of therapeutic antibody development has been motivated by the benefit to and their demand for human health. In particular, humanized transgenic antibody discovery platforms, combined with immunization, hybridoma fusion and/or single cell DNA sequencing are the most reliable and rapid methods for mining the human monoclonal antibodies. Human GPC3 protein is an oncofetal antigen, and it is highly expressed in most hepatocellular carcinomas and some types of squamous cell carcinomas. Currently, no fully human anti-GPC3 therapeutic antibodies have been reported and evaluated in extensive tumor tissues. Here, we utilized a new humanized transgenic mouse antibody discovery platform (CAMouse) that contains large V(D)J -regions and human gamma-constant regions of human immunoglobulin in authentic configurations to generate fully human anti-GPC3 antibodies. Our experiments resulted in four anti-GPC3 antibodies with high-specific binding and cytotoxicity to GPC3 positive cancer cells, and the antibody affinities are in the nanomolar range. Immunohistochemistry analysis demonstrated that these antibodies can recognize GPC3 protein on many types of solid tumors. In summary, the human anti-human GPC3 monoclonal antibodies described here are leading candidates for further preclinical studies of cancer therapy, further, the CAMouse platform is a robust tool for human therapeutic antibody discovery.
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Anticorpos Monoclonais/farmacologia , Glipicanas/antagonistas & inibidores , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glipicanas/imunologia , Glipicanas/metabolismo , Hepatite/metabolismo , Humanos , Intestino Delgado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos Transgênicos , Placenta/metabolismo , GravidezRESUMO
Simpson-Golabi-Behmel syndrome (SGBS) is a rare X-linked condition characterized by pre and postnatal overgrowth with visceral and skeletal abnormalities. The syndrome is caused mainly by mutations in the X-linked gene GPC3. Clinical presentation of SGBS in affected males is well defined, but there is a lack of knowledge about affected females, with very few reported cases. In total, eight female carriers with clinical expression of SGBS have been reported to date. In the present report, we describe the ninth patient and her family history. The interesting features of our female patient are the Wilms' tumor and the transfontanelar ultrasound findings. The patient's older sister, carrier of the same mutation, has minor facial dysmorphisms but no congenital anomalies and so far, no further clinical findings, as well as her mother and grandmother. There is a lesson to be learned from these rare cases, namely that SGBS may have a significant clinical expression in females, and therefore, screening should be considered in all patients with SGBS regardless of the sex or phenotypic severity.
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Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/genética , Estudos de Associação Genética , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Predisposição Genética para Doença , Gigantismo/diagnóstico , Gigantismo/genética , Glipicanas/genética , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação , Fenótipo , Alelos , Biópsia , Análise Mutacional de DNA , Fácies , Feminino , Genes Ligados ao Cromossomo X , Estudos de Associação Genética/métodos , Genótipo , Humanos , Lactente , Masculino , Linhagem , Inativação do Cromossomo XRESUMO
The glypican-3 (GPC3) receptor is a membrane protein that is highly expressed in tumor tissues but rarely expressed in the normal liver and can be used as a target for early diagnosis of hepatocellular carcinoma (HCC). Herein, we developed a GPC3-targeted 99mTc-labeled probe for SPECT imaging in HCC. 99mTc-HPG was rapidly radiosynthesized within 20 min with an excellent radiochemical purity (>98%), possessing good stability. Results from in vitro cell binding assays indicated that the binding specificity of 99mTc-HPG to GPC3-positive HepG2 cells was acceptable. For SPECT/CT imaging, the HepG2 tumors were clearly visualized with the highest tumor/muscle ratio (11.55 ± 0.54) at 1 h post-injection, and the tumor uptake of 99mTc-HPG reduced from 2.99 ± 0.15 to 1.17 ± 0.09% ID/g in the blocking study. Convenient preparation, excellent GPC3 specificity in HCC, rapid clearance from normal organs, and good biosafety profiles of 99mTc-HPG warrant further investigations for clinical translation.
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Carcinoma Hepatocelular/diagnóstico , Glipicanas/metabolismo , Neoplasias Hepáticas/diagnóstico , Compostos Radiofarmacêuticos/administração & dosagem , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Carcinoma Hepatocelular/patologia , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Imagem Molecular/métodos , Sondas Moleculares/administração & dosagem , Sondas Moleculares/farmacocinética , Compostos de Organotecnécio/administração & dosagem , Compostos de Organotecnécio/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glypican-3 (GPC3) is a serological biomarker for the diagnosis of Hepatocellular carcinoma (HCC), but it is a challenging task to develop a bioassay for determination of the trace GPC3 in serum. In this study, Bioluminescense immunoassay based on bifunctional nanobody-nanoluciferase fusion was developed with the ultra-sensitive feature to achieve this goal. First, nanobodies special against GPC-3 binder as biological recognition element were generated by immunization and phage display technology. Second, The best clone GPN2 was fused with nanoluciferase as a dual-functional immunoreagent to establish an ultra-sensitive bioluminescence enzyme immunoassay (BLEIA), which is 30 and 5 times more sensitive than the traditional colorimetric assay and fluorescent assay, respectively. The cross-reactivity analysis of BLEIA showed that there was no cross-reactivity with HCC related tumor markers AFP, CEA, CA19-9 and GPC1/GPC2. The limit of detection (LOD) of developed BLEIA was 1.5 ng/mL, which assured its application in the diagnosis of GPC3 in 94 serum samples. This study indicates that BLEIA based on nanobody-nanoluciferase fusion could be used as a useful tool for the diagnosis of HCC patients.
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BACKGROUND: This research was to develop a special method for enriching Circulating tumor cells (CTCs) of Hepatocellular carcinoma (HCC) by Glypican-3 immunoliposomes (GPC3-IML), and to analyze the correlation between the CTCs count and tumor malignancy, as well as to investigate the mutation characteristics of CTC-derived NGS. RESULTS: In this study characterization of physical parameters was performed with the preparation of GPC3-IML. CTCs in peripheral blood of HCC patients were further separated and identified. Immunofluorescence was used to identify CTCs for further counting. By this means, the correlation between CTCs count and clinicopathological features was analyzed, and the genetic mutation characteristics of NGS derived from CTCs were investigated and compared with that of tissue NGS. Results showed that compared with EpCAM and vimentin, GPC-3 had a stronger CTCs separation ability. There was a correlation between "positive" count of CTCs (≥ 5 PV-CTC per 7.5 ml blood) and BCLC stage (P = 0.055). The result of CTC-NGS was consistent with that of tissue-NGS in 60% cases, revealing that KMT2C was a common highly-frequent mutated gene. CONCLUSION: The combination of immunomagnetic separation of CTCs and anti-tumor marker identification technology can be regarded as a new technology of CTCs detection in peripheral blood of patients with HCC. Trial registration EHBHKY2020-k-024. Registered 17 August 2020-Retrospectively registered.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Carcinoma Hepatocelular/metabolismo , Glipicanas/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/sangue , Carboplatina/metabolismo , Carcinoma Hepatocelular/patologia , Ciclofosfamida/metabolismo , Molécula de Adesão da Célula Epitelial , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes , Tiotepa/metabolismo , Adulto JovemRESUMO
Simpson-Golabi-Behmel syndrome (SGBS) represents a rare X-linked recessive syndrome with prenatal and postnatal overgrowth, coarse facial features, congenital malformations, organomegaly and an increased risk of tumors. Mutations on the GPC3 gene, encoding the glypican-3 protein, have previously been shown to cause the disease. In this report, a 12-year-old Chinese boy was hospitalized in our institution for some clinical features of SGBS. His serum endocrine evaluation showed hormone level abnormalities, including high prolactin, high testosterone, high thyroid-stimulating hormone (TSH) levels, and low estradiol levels. Whole exome sequencing (WES) was performed in the patient for mutation analysis and a novel hemizygous mutation, c.185delT, p.(Leu62Cysfs*22), on the GPC3 gene, was identified. The mother was a heterozygous carrier. The SGBS patients might present with endocrine anomalies, which adds to the clinical heterogeneity of the disease. The novel GPC3 mutation c.185delT expands the mutational spectrum of the GPC3 gene.
RESUMO
The use of allogeneic, pluripotent stem-cell-derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator-initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) chimeric antigen receptor (CAR)-expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder-free production of CAR-expressing NK/ILC cells from CAR-transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8-3.6 × 106 iPSC within 7 weeks was 1.8-4.0 × 109 . These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN-γ) production against GPC3-expressing tumor cells. When the CAR-NK/ILC cells were injected into a GPC3-positive, ovarian-tumor-bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non-clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR-NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell-based immune cell cancer therapies.
Assuntos
Glipicanas/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Diferenciação Celular , Sobrevivência Celular , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Glipicanas/genética , Glipicanas/metabolismo , Humanos , Imunidade Inata , Imunoterapia Adotiva , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Interferon gama/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/transplante , Transfusão de Linfócitos , Linfócitos/citologia , Camundongos , Camundongos SCID , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismoRESUMO
The Xq26 locus has importance in human growth with multiple genes and regions playing important roles, which potentially leads to macrosomia or microsomia if disrupted. One region of Xq26.2 comprises the genes GPC3 and GPC4; deletion or duplication of this region has been recently been shown to result in overgrowth, specifically Simpson-Golabi-Behmel syndrome. We describe a male patient with two maternally inherited Xq26 microduplications; the first was 0.8 Mb at Xq26.2 affecting only GPC3 and GPC4, and the second, a distal 0.6 Mb duplication at Xq26.3 affecting seven genes. Rather than having Simpson-Golabi-Behmel syndrome, our patient had microcephaly and undergrowth, with development that was within normal limits at 25 months of age. This finding suggests that the molecular pathway leading to overgrowth secondary to GPC3/GPC4 haploinsufficiency can be overpowered by a disruption to the distal Xq26.3 region. Of the genes in that region, we propose that SLC9A6 is the most likely to play an important role as mutations in this gene lead to Christianson syndrome, in which patients may have microcephaly and weight loss.