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1.
EMBO J ; 40(19): e108375, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34375000

RESUMO

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.


Assuntos
Receptores Mitogênicos/metabolismo , SARS-CoV-2/metabolismo , Animais , Sítios de Ligação/fisiologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Glicosilação , Células HEK293 , Humanos , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica/fisiologia , Células Vero , Internalização do Vírus
2.
Cell Rep Methods ; 4(4): 100728, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38492569

RESUMO

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast, CAR T cell treatment of solid tumors is associated with several challenges, in particular the expression of most tumor-associated antigens at lower levels in vital organs, resulting in on-target/off-tumor toxicities. Thus, innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands, we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show, in several experimental systems, that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers.


Assuntos
Receptores ErbB , Receptores de Antígenos Quiméricos , Linfócitos T , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunoterapia Adotiva/métodos , Animais , Neoplasias/imunologia , Neoplasias/terapia , Linhagem Celular Tumoral , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Camundongos
3.
Sci Rep ; 13(1): 23024, 2023 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-38155191

RESUMO

The majority of approved CAR T cell products are based on the FMC63-scFv directed against CD19. Surprisingly, although antigen binding affinity is a major determinant for CAR function, the affinity of the benchmark FMC63-scFv has not been unambiguously determined. That is, a wide range of affinities have been reported in literature, differing by more than 100-fold. Using a range of techniques, we demonstrate that suboptimal experimental designs can cause artefacts that lead to over- or underestimation of the affinity. To minimize these artefacts, we performed SPR with strictly monomeric and correctly folded soluble CD19, yielding an FMC63-scFv affinity of 2-6 nM. Together, apart from analyzing the FMC63-scFv affinity under optimized conditions, we also provide potential explanations for the wide range of published affinities. We expect that this study will be highly valuable for interpretations of CAR affinity-function relationships, as well as for the design of future CAR T cell generations.


Assuntos
Imunoterapia Adotiva , Linfócitos T , Imunoterapia Adotiva/métodos , Antígenos CD19
4.
Materials (Basel) ; 15(18)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36143674

RESUMO

In the present work, an ultrasonic, an induction, and a through transmission laser welding were compared to join carbon fibre reinforced polyetheretherketone (CF/PEEK) composites. The advantages and drawbacks of each process are discussed, as well as the material properties required to fit each process. CF/PEEK plates were consolidated at 395 °C with an unidirectional sequence and cross-stacking ply orientation. In some configurations, a polyetherimide (PEI) layer or substrate was used. The thermal, mechanical, and optical properties of the materials were measured to highlight the specific properties required for each process. The drying conditions were defined as 150 °C during at least 8 h for PEI and 24 h for CF/PEEK to avoid defects due to water. The optical transmission factor of PEI is above 40% which makes it suitable for through transmission laser welding. The thermal conductivity of CF/PEEK is at most 55 W·(m·K)-1, which allows it to weld by induction without a metallic susceptor. Ultrasonic welding is the most versatile process as it does not necessitate any specific properties. Then, the mechanical resistance of the welds was measured by single lap shear. For CF/PEEK on CF/PEEK, the maximum lap shear strength (LSS) of 28.6 MPa was reached for a joint obtained by ultrasonic welding, while an induction one brought 17.6 MPa. The maximum LSS of 15.2 MPa was obtained for PEI on CF/PEEK assemblies by laser welding. Finally, interfacial resistances were correlated to the fracture modes through observations of the fractured surfaces. CF/PEEK on CF/PEEK joints resulted in mixed cohesive/adhesive failure at the interface and within the inner layers of both substrates. This study presents a guideline to select the suitable welding process when assembling composites for the aerospace industry.

5.
Biotechnol J ; 17(5): e2100422, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35078277

RESUMO

The receptor binding domain (RBD) of the SARS-CoV-2 spike (S)-protein is a prime target of virus-neutralizing antibodies present in convalescent sera of COVID-19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293-6E cells towards a stable homogeneous preparation and addressed its O-glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319-541) sequence. The authors found that an intact O-glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead-based and enzyme-linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS-CoV-2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS-CoV-2 tests and inform the design of RBD-based vaccine candidates.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/terapia , Cisteína , Células HEK293 , Humanos , Imunização Passiva , Mamíferos , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Soroterapia para COVID-19
6.
Nat Commun ; 13(1): 7926, 2022 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-36566234

RESUMO

Recent waves of COVID-19 correlate with the emergence of the Delta and the Omicron variant. We report that the Spike trimer acts as a highly dynamic molecular caliper, thereby forming up to three tight bonds through its RBDs with ACE2 expressed on the cell surface. The Spike of both Delta and Omicron (B.1.1.529) Variant enhance and markedly prolong viral attachment to the host cell receptor ACE2, as opposed to the early Wuhan-1 isolate. Delta Spike shows rapid binding of all three Spike RBDs to three different ACE2 molecules with considerably increased bond lifetime when compared to the reference strain, thereby significantly amplifying avidity. Intriguingly, Omicron (B.1.1.529) Spike displays less multivalent bindings to ACE2 molecules, yet with a ten time longer bond lifetime than Delta. Delta and Omicron (B.1.1.529) Spike variants enhance and prolong viral attachment to the host, which likely not only increases the rate of viral uptake, but also enhances the resistance of the variants against host-cell detachment by shear forces such as airflow, mucus or blood flow. We uncover distinct binding mechanisms and strategies at single-molecule resolution, employed by circulating SARS-CoV-2 variants to enhance infectivity and viral transmission.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Imagem Individual de Molécula , Glicoproteína da Espícula de Coronavírus , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral
7.
ACS Synth Biol ; 10(5): 1184-1198, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33843201

RESUMO

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.


Assuntos
Substituição de Aminoácidos , Antígenos CD19/genética , Evolução Molecular Direcionada/métodos , Imunoterapia Adotiva/métodos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Aminoácidos/genética , Anticorpos Monoclonais/imunologia , Antígenos CD19/química , Antígenos CD19/imunologia , Células HEK293 , Humanos , Linfoma Difuso de Grandes Células B/sangue , Proteínas Mutantes , Mutação , Domínios Proteicos/imunologia , Dobramento de Proteína , Estabilidade Proteica , Receptores de Antígenos Quiméricos/genética
8.
Front Plant Sci ; 12: 689104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34211491

RESUMO

Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.

9.
Biotechnol J ; 16(6): e2000566, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33481336

RESUMO

Human angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019. Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS-CoV-2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2-fragment crystallizable (Fc) variant in glycoengineered Nicotiana benthamiana. Our data reveal that the produced dimeric ACE2-Fc variant is glycosylated with mainly complex human-type N-glycans and functional with regard to enzyme activity, affinity to the SARS-CoV-2 receptor-binding domain, and wild-type virus neutralization.


Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Humanos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Glicoproteína da Espícula de Coronavírus , Nicotiana/genética , Nicotiana/metabolismo
10.
Elife ; 102021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34927585

RESUMO

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Simulação de Dinâmica Molecular , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , COVID-19/prevenção & controle , COVID-19/virologia , Glicosilação , Humanos , Polissacarídeos/química , Ligação Proteica , Engenharia de Proteínas , Receptores Virais/química , Receptores Virais/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
11.
Front Plant Sci ; 12: 747500, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34646292

RESUMO

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, N-glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered Nicotiana benthamiana leaves and characterized the impact of different plant-specific N-glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying ß1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average.

12.
EBioMedicine ; 67: 103348, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33906067

RESUMO

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sítios de Ligação , Células CHO , COVID-19/imunologia , Cricetulus , Diagnóstico Precoce , Células HEK293 , Humanos , Imunoglobulina G/sangue , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
14.
Anal Chim Acta ; 614(1): 63-70, 2008 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-18405682

RESUMO

In a first step towards chemical sensors using molecular imprinted materials, the complexing characteristics of diethyl 4-nitrobenzylphosphonate, an organophosphate pesticide analogue, have been studied. Two molecules have been assessed as potential interacting moieties, specifically a fluoroalcohol and an aromatic acid. The interactions have been first characterized by regular methods, such as 1H, 31P NMR and IR spectroscopy. These showed a stoichiometry 1/1 for both complexes and association constants, respectively, close to 40+/-10 and 12+/-2 M(-1). In a second step, isothermal titration calorimetry was used and a method was developed to obtain low-association constants. The association constant could be obtained for the fluoroalcohol ligand and was found equal to 63+/-0.7 M(-1). For the acidic molecule, an appropriate model could not be found, preventing the evaluation of this constant.


Assuntos
Compostos de Benzil/análise , Compostos Organofosforados/análise , Praguicidas/análise , Espectroscopia de Ressonância Magnética , Espectrofotometria Infravermelho
15.
Anal Chim Acta ; 591(1): 1-6, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17456417

RESUMO

Several liquid crystalline imprinted materials have been synthesized from polysiloxanes or polyacrylates bearing mesogenic side-chains and low contents of cross-linkers. They were imprinted by various achiral or chiral templates, then tested for molecular recognition or assessed as specific catalysts. All mesogenic imprinted networks exhibit a much higher affinity towards the template than non-imprinted networks. On the other hand, the molecular trapping capacity was shown to be much greater than that of most of the previously studied non-mesomorphous systems. Moreover, it was shown that mesomorphic order provides significant enhancement to the bonding between the template and the liquid crystalline network and reinforces the shape memory of the imprinted cavities. Some of these materials were used to catalyze the isomerization of benzisoxazole. They exhibited an acceleration effect close to 100 between imprinted sites and non-imprinted ones. Lastly, cholesteric networks, that were imprinted by a chiral template, showed good properties for the enantiomer separation leading to an enantiomeric excess of 35% and a capacity of around 1 mmol g(-1).

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