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Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.
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Anticorpos Biespecíficos , Proteólise , Humanos , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/imunologia , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Proteínas que Contêm BromodomínioRESUMO
Purpose: As capsule elongation is assumed to weaken the static stability of the shoulder joint, the purpose of this biomechanical study was to demonstrate that capsule elongation occurs immediately after a first-time shoulder dislocation and not just after recurrent dislocation events. We hypothesize an increment in joint clearance due to joint capsule elongation after a first-time dislocation. Methods: An experimental in-vitro study was conducted on 6 paired fresh frozen human shoulders (4 females; 2 males; 12 specimen) with a mean age of 80 (Range 67-89) years. The shoulder joint with the articular capsule was exposed and an inferior static tension force of 2.5 N was applied to the humerus prior to dislocation. Next, the humeral head was dislocated and was then immediately reduced back into the start position. The joint gap as well as joint capsule deformation was assessed using optical techniques. Results: The radiographic joint gap increased from 13.7 ± 6.9 mm (prior to dislocation) to 18.1 ± 6.5 mm (post dislocation) (p < .001). The increase in joint clearance was 4.4 mm. The joint capsule elongated from 5.9 ± 0.005 % (prior to dislocation) to 9.4 ± 0.007 % (post dislocation) (p < .001). The mean increase in joint capsule elongation was 3.5 %. Conclusions: Capsule elongation was observed immediately after a simulated first-time shoulder dislocation in an in-vitro model of elderly human cadavers. It might therefore not only be a phenomenon of recurrent dislocation events.
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BACKGROUND: To reduce hardware-related complications in coracoid graft fixation to the anterior aspect of the glenoid, a metal-free Latarjet technique was recently introduced. The aim of this study was to compare the primary stability of a classic Latarjet procedure with 2 metal screws to a novel metal-free, all-suture cerclage method. It is hypothesized that fixation of the coracoid graft with 2 malleolar screws will provide higher primary stability compared with an all-suture cerclage technique. METHODS: This biomechanical in vitro study was conducted on 12 fresh-frozen cadaveric shoulders (6 matched pairs) with a mean donor age of 80 years (range, 67-89 years). Coracoid graft fixation was performed using a recently introduced all-suture cerclage technique (group A) or a classic Latarjet technique with two 4.5-mm malleolar screws (group B). The conjoint tendon was loaded with a static force of 10 N to simulate the sling effect. Graft loading with a probe head consisted of 6 ascending load levels (10-50 N, 10-100 N, 10-150 N, 10-200 N, 10-250 N, and 10-300 N) with 100 cycles each at 1 Hz. Relative motion of the bone graft to the glenoid was measured using an optic 3-dimensional system. RESULTS: While loading the conjoint tendon with 10 N, no difference in mean displacement of the bone-graft was found between both groups (P = .144). During cyclic loading, a significant difference in relative displacement for both groups was already detected in load level 1 (group A: 2398.8 µm vs. group B: 125.7 µm; P = .024), and this trend continued with the following load levels (P < .05). DISCUSSION AND CONCLUSION: The study demonstrated that the innovative metal-free, all-suture cerclage fixation technique results in higher micromotion than the classic coracoid graft fixation with 2 malleolar screws. According to the present biomechanical investigation, shoulders treated with a metal-free all-suture cerclage technique might need adapted rehabilitation protocols to protect the construct and allow for graft healing.
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Bursite , Instabilidade Articular , Luxação do Ombro , Articulação do Ombro , Humanos , Idoso , Idoso de 80 Anos ou mais , Articulação do Ombro/cirurgia , Transplante Ósseo , Escápula/cirurgia , Luxação do Ombro/cirurgia , Suturas , Fenômenos Biomecânicos , Instabilidade Articular/cirurgiaRESUMO
A crucial design feature for the therapeutic success of antibody-drug conjugates (ADCs) is the linker that connects the antibody with the drug. Linkers must be stable in circulation and efficiently release the drug inside the target cell, thereby having a fundamental impact on ADC pharmacokinetics and efficacy. The variety of enzymatically cleavable linkers applied in ADCs is limited, and some are believed to be associated with unwanted side effects due to the expression of cleavage-mediating enzymes in nonmalignant cells. Based on a bioinformatic screen of lysosomal enzymes, we identified α-l-iduronidase (IduA) as an interesting candidate for ADC linker cleavage because of its low expression in normal tissues and its overexpression in several tumor types. In the present study, we report a novel IduA-cleavable ADC linker using exatecan and duocarmycin as payloads. We showed the functionality of our linker system in cleavage assays using recombinant IduA or cell lysates and compared it to established ADC linkers. Subsequently, we coupled iduronide-exatecan via interchain cysteines or iduronide-duocarmycin via microbial transglutaminase (mTG) to an anti-CEACAM5 (aCEA5) antibody. The generated iduronide-exatecan ADC showed high serum stability and similar target-dependent tumor cell killing in the subnanomolar range but reduced toxicity on nonmalignant cells compared to an analogous cathepsin B-activatable valine-citrulline-exatecan ADC. Finally, in vivo antitumor activity could be demonstrated for an IduA-cleavable duocarmycin ADC. The presented results emphasize the potential of iduronide linkers for ADC development and represent a tool for further balancing out tumor selectivity and safety.
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Antineoplásicos , Imunoconjugados , Imunoconjugados/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/metabolismo , Iduronidase , Duocarmicinas , Anticorpos Monoclonais , Linhagem Celular TumoralRESUMO
BACKGROUND: Modularity in revision THA (RTHA) has become accepted during the last three decades. Nevertheless, specific risks of modularity of current revision devices such as breakage of taper junctions occur during follow-up. Data reporting failure rates are predominantly given by the manufacturers but independent data acquisition is missing so far. QUESTIONS/PURPOSES: 1. What time-related risk of breakage of taper junction between neck and body of an established modular revision device can be expected in a consecutive single institutional series and a mid-term follow-up? 2. Are there specific factors influencing breakage in this cohort? MATERIALS AND METHODS: A retrospective analysis was performed of a consecutive series of 89 cases after femoral revision using a tapered modular revision stem. Mean follow-up period was 7.1 (range: 3.0-13.7) years. Breakage of stem as failure criteria of the implant was investigated with a Kaplan-Meier analysis. RESULTS: Breakage of taper junctions occurred in four patients during follow-up showing a time-depending implant survival of 94.2 (95% CI: 88.6-100%) after 13.7 years. Implant survival of stems with lateralized necks of 87.4 (95% CI: 75.6-100%) after 13.7 years was significantly lower compared to the standard offset variant with 100% after 13.5 years (log rank test p = 0.0283). Chi square test also revealed a significantly higher risk of breakage of lateralized necks compared to standard offset pieces (p = 0.0141). Three of four patients were obese with a mean BMI of 37.9 kg/m2. Grade of obesity (grade 1 or higher) had significant influence on risk of breakage. Survival of the implant was significantly lower in obese patients with at least grade 1 obesity compared to patients with a BMI < 30 kg/m2 (82.9 (95% CI: 64.9-100%) after 11.6 years vs. 98.4 (95% CI: 95.3-100%) after 13.7 years; log-rank p = 0.0327). CONCLUSIONS: Cumulative risk for failure of taper junctions was high in this consecutive single institutional cohort and may further increase during follow-up. As independent data acquisition in registries is missing, failure rate may be higher than reported data of the manufacturers. The use of lateralized offset necks in obese patients of at least grade 1 obesity showed a significantly higher risk of breakage. The use of monobloc revision devices may be an option, but randomized control trials are currently missing to establish standardized treatment protocols considering individual risks for both monobloc and/or modular implants.
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PURPOSE: Reconstruction of the medial patellofemoral ligament (MPFL-R) with nonresorbable suture tape (FiberTape®, FT) is becoming popular. Patella-side fixation of the FT can be performed with suture anchors or via soft-tissue fixation. The aim of this study was to investigate whether patella-side soft-tissue fixation can achieve equivalent primary stability compared to suture-anchor fixation. METHODS: In ten human, fresh-frozen knee joint specimens (m/f 6/4; age 74 ± 9 a), the MPFL was identified and dissected near the femoral insertion site. In five knee joints, the MPFL-R using FT was performed with soft-tissue fixation at the patella (study group; SG), and in five knee joints, the FT was fixed via suture anchors (control group, CG). All reconstructions were evaluated until load to failure of the patella-side fixation with a displacement rate of 200 mm/min. RESULTS: The mean maximum load to failure in the SG was 395.3 ± 57.9 N. All reconstructions failed by complete tearing off the medial patellar retinaculum from its medial patellar margin, but fixation of the FT remained stable. In the CG, the mean maximum load to failure was 239.4 ± 54.5 N and was significantly different compared to the SG (p = 0.04). All reconstructions failed via pullout of the suture anchors. Stiffness and elongation did not differ between the groups, and no failure of the FT was observed in any of the specimens. CONCLUSION: Primary stability of soft-tissue MPFL-R using FT was superior to suture-anchor fixation. Both fixation techniques provided sufficient primary stability, superior to previously reported native MPFL tensile strengths. MPFL-R with FT could be a possible alternative procedure for MPFL-R, eliminating potential complications due to autologous tendon graft harvesting.
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Articulação Patelofemoral , Âncoras de Sutura , Humanos , Idoso , Idoso de 80 Anos ou mais , Articulação Patelofemoral/cirurgia , Fixação de Tecidos , Patela/cirurgia , Articulação do Joelho , Ligamentos Articulares/cirurgia , SuturasRESUMO
Substituted diphenylthioureas (DPTUs) are efficient hydrogen-bonding organo-catalysts, and substitution of DPTUs has been shown to greatly affect catalytic activity. Yet, both the conformation of DPTUs in solution and the conformation and hydrogen-bonded motifs within catalytically active intermediates, pertinent to their mode of activation, have remained elusive. By combining linear and ultrafast vibrational spectroscopy with spectroscopic simulations and calculations, we show that different conformational states of thioureas give rise to distinctively different N-H stretching bands in the infrared spectra. In the absence of hydrogen-bond-accepting substrates, we show that vibrational structure and dynamics are highly sensitive to the substitution of DPTUs with CF3 groups and to the interaction with the solvent environment, allowing for disentangling the different conformational states. In contrast to bare diphenylthiourea (0CF-DPTU), we find the catalytically superior CF3-substituted DPTU (4CF-DPTU) to favor the trans-trans conformation in solution, allowing for donating two hydrogen bonds to the reactive substrate. In the presence of a prototypical substrate, DPTUs in trans-trans conformation hydrogen bond to the substrate's C=O group, as evidenced by a red-shift of the N-H vibration. Yet, our time-resolved infrared experiments indicate that only one N-H group forms a strong hydrogen bond to the carbonyl moiety, while thiourea's second N-H group only weakly interacts with the substrate. Our data indicate that hydrogen-bond exchange between these N-H groups occurs on the timescale of a few picoseconds for 0CF-DPTU and is significantly accelerated upon CF3 substitution. Our results highlight the subtle interplay between conformational equilibria, bonding states, and bonding lifetimes in reactive intermediates in thiourea catalysis, which help rationalize their catalytic activity.
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The exposition of cancer cells to cytotoxic doses of payload is fundamental for the therapeutic efficacy of antibody drug conjugates (ADCs) in solid cancers. To maximize payload exposure, tissue penetration can be increased by utilizing smaller-sized drug conjugates which distribute deeper into the tumor. Our group recently explored small human epidermal growth factor receptor 2 (HER2) targeting Fc antigen binding fragments (Fcabs) for ADC applications in a feasibility study. Here, we expand this concept using epidermal growth factor receptor (EGFR) targeting Fcabs for the generation of site-specific auristatin-based drug conjugates. In contrast to HER2-targeting Fcabs, we identified novel conjugation sites in the EGFR-targeting Fcab scaffold that allowed for higher DAR enzymatic conjugation. We demonstrate feasibility of resultant EGFR-targeting Fcab-drug conjugates that retain binding to half-life prolonging neonatal Fc receptor (FcRn) and EGFR and show high serum stability as well as target receptor mediated cell killing at sub-nanomolar concentrations. Our results emphasize the applicability of the Fcab format for the generation of drug conjugates designed for increased penetration of solid tumors and potential FcRn-driven antibody-like pharmacokinetics.
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Antineoplásicos , Imunoconjugados , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Recém-Nascido , Ligação ProteicaRESUMO
The goal of this study is to evaluate the primary stability of a cementless augment-and-modular-cage system with and without the addition of cranial straps in a standardized in vitro setting. As the surrogate parameter for the evaluation of primary stability, the measurement of relative motion between the implant components themselves and the bone will be used. Acetabular revision components with a trabecular titanium augment in combination with a large fourth-generation composite left hemipelvis were assembled. These constructs were divided into two groups with (S) and without cranial straps (nS). A total of 1000 cycles was applied at each of three load levels. Relative movements (RM) between the components were measured. Load levels display a significant effect on the amount of RM at all interfaces except between shell/augment. The group assignment appears to have an effect on RM due to significantly differing means at all interfaces. Between bone/shell RM increased as load increased. NS displayed significantly more RM than S. Between shell/augment RM remained constant as load increased. Between shell/cup S showed more RM than nS while both groups' RM increased with load. We conclude a significant increase of primary stability between the shell and the bone through the addition of cranial straps. Relative motion between components (shell/cup) increases through the addition of cranial straps. A clinical impact of this finding is uncertain and requires further investigation. Finally, the cementless fixation of the augment against the rim-portion of the shell appears stable and compares favorably to prior investigation of different fixation techniques.
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With the increasing importance and complexity of data pipelines, data quality became one of the key challenges in modern software applications. The importance of data quality has been recognized beyond the field of data engineering and database management systems (DBMSs). Also, for machine learning (ML) applications, high data quality standards are crucial to ensure robust predictive performance and responsible usage of automated decision making. One of the most frequent data quality problems is missing values. Incomplete datasets can break data pipelines and can have a devastating impact on downstream ML applications when not detected. While statisticians and, more recently, ML researchers have introduced a variety of approaches to impute missing values, comprehensive benchmarks comparing classical and modern imputation approaches under fair and realistic conditions are underrepresented. Here, we aim to fill this gap. We conduct a comprehensive suite of experiments on a large number of datasets with heterogeneous data and realistic missingness conditions, comparing both novel deep learning approaches and classical ML imputation methods when either only test or train and test data are affected by missing data. Each imputation method is evaluated regarding the imputation quality and the impact imputation has on a downstream ML task. Our results provide valuable insights into the performance of a variety of imputation methods under realistic conditions. We hope that our results help researchers and engineers to guide their data preprocessing method selection for automated data quality improvement.
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Fragment crystallizable (Fc) antigen binding fragments (Fcabs) represent a novel antibody format comprising a homodimeric Fc region with an engineered antigen binding site. In contrast to their full-length antibody offspring, Fcabs combine Fc-domain-mediated and antigen binding functions at only one-third of the size. Their reduced size is accompanied by elevated tissue penetration capabilities, which is an attractive feature for the treatment of solid tumors. In the present study, we explored for the first time Fcabs as a novel scaffold for antibody-drug conjugates (ADCs). As model, various HER2-targeting Fcab variants coupled to a pH-sensitive dye were used in internalization experiments. A selective binding on HER2-expressing tumor cells and receptor-mediated endocytosis could be confirmed for selected variants, indicating that these Fcabs meet the basic prerequisite for an ADC approach. Subsequently, Fcabs were site-specifically coupled to cytotoxic monomethyl auristatin E yielding homogeneous conjugates. The conjugates retained HER2 and FcRn binding behavior of the parent Fcabs, showed a selective in vitro cell killing and conjugation site-dependent serum stability. Moreover, Fcab conjugates showed elevated penetration in a spheroid model, compared to their full-length antibody and Trastuzumab counterparts. Altogether, the presented results emphasize the potential of Fcabs as a novel scaffold for targeted drug delivery in solid cancers and pave the way for future in vivo translation.
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Sistemas de Liberação de Medicamentos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Anticorpos Monoclonais Humanizados/química , Sítios de Ligação , Linhagem Celular Tumoral , Corantes Fluorescentes , Humanos , Modelos Moleculares , Proteínas de Neoplasias , Ligação Proteica , Receptor ErbB-2 , Esferoides Celulares , TrastuzumabRESUMO
When it comes to total hip replacements, choosing the suitable material combination is of clinical relevance. The present review article examines the technical differences in wear and corrosion of the relevant material combinations of ceramics, metals, ceramized metals and various types of polyethylene. The material characteristics, which were often tested under standardized conditions in the laboratory, are compared with clinical results on the basis of evidence-graded clinical studies and on the basis of register studies. This article thus represents an up-to-date snapshot of the expectations and actual clinical outcomes of the present choice of material combinations. It shows that some tendencies from the field of materials research, e.g. with regard to cross-linked polyethylene, coincide with observations from practical clinical experience, while for other materials, a proven technical superiority has not yet been confirmed as an evident advantage in clinical practice.
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Artroplastia de Quadril , Prótese de Quadril , Humanos , Polietileno , Desenho de Prótese , Falha de PróteseRESUMO
AIMS: Left ventricular (LV) dysfunction in viral myocarditis is attributed to myocardial inflammation and fibrosis, inducing acute and long-time cardiac damage. Interventions are not established. On the basis of the link between inflammation, fibrosis, aldosterone, and extracellular matrix regulation, we aimed to investigate the effect of an early intervention with the mineralocorticoid receptor antagonist (MRA) eplerenone on cardiac remodelling in a murine model of persistent coxsackievirus B3 (CVB3)-induced myocarditis. METHODS AND RESULTS: SWR/J mice were infected with 5 × 104 plaque-forming units of CVB3 (Nancy strain) and daily treated either with eplerenone (200 mg/kg body weight) or with placebo starting from Day 1. At Day 8 or 28 post infection, mice were haemodynamically characterized and subsequently sacrificed for immunohistological and molecular biology analyses. Eplerenone did not influence CVB3 load. Already at Day 8, 1.8-fold (P < 0.05), 1.4-fold (P < 0.05), 3.2-fold (P < 0.01), and 2.1-fold (P < 0.001) reduction in LV intercellular adhesion molecule 1 expression, presence of monocytes/macrophages, oxidative stress, and apoptosis, respectively, was observed in eplerenone-treated vs. untreated CVB3-infected mice. In vitro, eplerenone led to 1.4-fold (P < 0.01) and 1.2-fold (P < 0.01) less CVB3-induced cardiomyocyte oxidative stress and apoptosis. Furthermore, collagen production was 1.1-fold (P < 0.05) decreased in cardiac fibroblasts cultured with medium of eplerenone-treated vs. untreated CVB3-infected HL-1 cardiomyocytes. These ameliorations were in vivo translated into prevention of cardiac fibrosis, as shown by 1.4-fold (P < 0.01) and 2.1-fold (P < 0.001) lower collagen content in the LV of eplerenone-treated vs. untreated CVB3-infected mice at Days 8 and 28, respectively. This resulted in an early and long-lasting improvement of LV dimension and function, as indicated by reduced LV end-systolic volume and end-diastolic volume, and an increase in LV contractility (dP/dtmax ) and LV relaxation (dP/dtmin ), respectively (P < 0.05). CONCLUSIONS: Early intervention with the MRA eplerenone modulates the acute host and defence reaction and prevents cardiac disease progression in experimental CVB3-induced myocarditis without aggravation of viral load. The findings advocate for an initiation of therapy of viral myocarditis as early as possible, even before the onset of inflammation-induced myocardial dysfunction. This may also have implications for coronavirus disease-19 therapy.
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Fibrose Endomiocárdica/prevenção & controle , Enterovirus Humano B/patogenicidade , Eplerenona/farmacologia , Miocardite/tratamento farmacológico , Miocardite/virologia , Disfunção Ventricular Esquerda/virologia , Análise de Variância , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Progressão da Doença , Fibrose Endomiocárdica/patologia , Imuno-Histoquímica , Masculino , Metaloproteinases da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Miocardite/prevenção & controle , Distribuição Aleatória , Valores de Referência , Resultado do Tratamento , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Frequently occurring damage, as well as elevated blood metal ion levels, are reported in relation to a tumor and revision system for total knee arthroplasty (TKA), which applies a rotating hinge coupling with a metal-on-metal (MoM) articulation. As the patient collective for this specific system is small, there is no data on wear generated from the couplings. In this study, wear volume and influencing parameters were investigated at 44 retrieved TKAs with MoM couplings. A scoring system rating frequently occurring abrasive wear between 0 (no wear) and 3 (distinct wear) was established. The wear score was correlated to time in vivo, bone resection length, patient weight and polyethylene inlay damage. Volumetric wear was estimated applying coordinate measurements. An elevated wear score of two or higher was found in 43% of cases. The mean wear rate accounted to 7.8 mm3/year. The main influencing coefficient for the extent of wear is time in vivo. We found a tendency for higher wear scores with higher inlay degradation scores. Patient weight and bone resection length did not impact coupling wear. Assessment of wear damage by a semi-quantitative scoring system has proven to be a reliable option for non-destructive coupling evaluation. The generated wear volume is high.
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The antibody repertoire of cartilaginous fish comprises an additional heavy-chain-only antibody isotype that is referred to as IgNAR (immunoglobulin novel antigen receptor). Its antigen-binding site consists of one single domain (vNAR) that is reportedly able to engage a respective antigen with affinities similar to those achieved by conventional antibodies. While vNAR domains offer a reduced size, which is often favorable for applications in a therapeutic as well as a biotechnological setup, they also exhibit a high physicochemical stability. Together with their ability to target difficult-to-address antigens such as virus particles or toxins, these shark-derived antibody domains seem to be predestined as tools for biotechnological and diagnostic applications. In the following chapter, we will describe the isolation of anti-idiotypic vNAR domains targeting monoclonal antibody paratopes from semi-synthetic, yeast-displayed libraries. Anti-idiotypic vNAR variants could be employed for the characterization of antibody-based therapeutics (such as antibody-drug conjugates) or as positive controls in immunogenicity assays. Peculiarly, when using semi-synthetic vNAR libraries, we found that it is not necessary to deplete the libraries using unrelated antibody targets, which enables a fast and facile screening procedure that exclusively delivers anti-idiotypic binders.
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Anticorpos Anti-Idiotípicos , Proteínas de Peixes , Biblioteca de Peptídeos , Tubarões , Anticorpos de Cadeia Única , Animais , Anticorpos Anti-Idiotípicos/química , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Domínios Proteicos , Tubarões/genética , Tubarões/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.
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Anticorpos Monoclonais , Citometria de Fluxo , Imunoglobulina G , Biblioteca de Peptídeos , Receptores de Antígenos de Linfócitos B , Saccharomyces cerevisiae , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Ratos , Ratos Transgênicos , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Anti-hapten antibodies are widely used as specific immunochemical detection tools in a variety of clinical and environmental analyses. The sensitivity, however, is limited due to the resulting antibody affinities to the haptens which, in turn, leads to a high demand for specific affinity reagents. A well-established path for the generation of high-affinity antibodies is the immunization of animals with the target antigen. However, the generation of anti-hapten antibodies via immunization remains challenging as small molecule haptens usually possess low immunogenicity and, therefore, must be coupled to an immunogenic and high molecular weight carrier to provoke an immune response.Consequently, antibodies are primarily raised against the carrier molecule or structural features of the hapten-linker fused to the carrier protein. This turns the generation of antibodies which bind exclusively to the hapten structure into a search for the needle in a haystack. In the following chapter, we describe how yeast surface display and high-throughput fluorescence-activated cell sorting can be used to isolate anti-hapten antibodies from a large, yeast-displayed B-cell receptor gene library derived from immunized animals. For this, we describe in detail the preparation of protein-hapten conjugates, the immunization procedure, and the subsequent screening process. Moreover, we provide a simple flow cytometry protocol that allows for a rapid analysis of the enriched clones toward free hapten binding.
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Anticorpos Monoclonais , Haptenos , Biblioteca de Peptídeos , Receptores de Antígenos de Linfócitos B , Saccharomyces cerevisiae , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Camelídeos Americanos , Galinhas , Haptenos/química , Haptenos/imunologia , Camundongos , Coelhos , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , OvinosRESUMO
A simple and efficient method to synthesize the immunogenic glycolipid BbGL1 is introduced. Two simple steps were required to obtain the desired product in good yield. First, a highly efficient glycosylation of cholesterol using galactosyl trichloroacetimidate as a donor was performed to produce cholesteryl-ß-d-galactoside. Finally, an efficient palmitoylation on the C6-OH of the galactose of the synthesized saponin using sym-collidine and acyl chloride under microwave heating that produced BbGL1 in good yield. The procedure is a convenient and cheaper alternative to the reported procedures allowing a rapid preparation of multiple analogs and conjugates.
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Glicolipídeos/síntese química , Glicolipídeos/imunologia , Saponinas/síntese química , Saponinas/imunologia , Glicolipídeos/química , Glicosilação , Conformação Molecular , Saponinas/químicaRESUMO
A series of novel N1-(4,5-diazafluoren-9-yliden)-N2-glycopyranosyl hydrazines was prepared in synthetically useful yields by treatment of 9H-4,5-diazafluoren-9-hydrazone with different unprotected monosaccharides. The reactions with the monosaccharides tested afforded stereoselectively, and exclusively, cyclic derivatives, whose structures correspond to N-ß-glycopyranosyl hydrazones except for the d-arabinose derivative that agrees with the α-anomer. Several copper(II) complexes having a 2:1 ligand to metal mole ratio were also prepared. The metal complexes can bind DNA sequences and preferentially stabilize G-quadruplex DNA structures over dsDNA. The fucose, rhamnose and deoxyglucose copper(II) complexes exhibited a cytotoxic activity against cultured HeLa and PC3 tumor cells comparable to other metal complexes normally used for chemotherapeutic purposes, such as cisplatin.
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Antineoplásicos/química , Antineoplásicos/farmacologia , Fluorenos/química , Fluorenos/farmacologia , Hidrazonas/química , Hidrazonas/farmacologia , Antineoplásicos/síntese química , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Fluorenos/síntese química , Glicoconjugados/síntese química , Glicoconjugados/química , Glicoconjugados/farmacologia , Células HeLa , Humanos , Hidrazonas/síntese química , Neoplasias/tratamento farmacológico , Células PC-3RESUMO
ViscumTT, a whole mistletoe preparation, has shown synergistic induction of apoptosis in several pediatric tumor entities. High therapeutic potential has previously been observed in Ewing's sarcoma, rhabdomyosarcoma, ALL and AML. In this study, we analyzed modulatory effects on the cell cycle by viscumTT in three osteosarcoma cell lines with various TP53 statuses. ViscumTT treatment induced G1 arrest in TP53 wild-type and null-mutant cells, but S arrest in TP53 mutant cells. Blockage of G1/S transition was accompanied by down-regulation of the key regulators CDK4, CCND1, CDK2, CCNE, CCNA. However, investigations on the transcriptional level revealed secondary TP53 participation. Cell cycle arrest was predominantly mediated by transcriptionally increased expression of GADD45A and CDKN1A and decreased SKP2 levels. Enhanced CDKN1A and GADD45A expression further played a role in viscumTT-induced apoptosis with involvement of stress-induced MAPK8 and inactivation of MAPK1/3. Furthermore, viscumTT inhibited the pro-survival pathway STAT3 by dephosphorylation of the two sites, Tyr705 and Ser727, by down-regulation of total STAT3 and its direct downstream targets BIRC5 and C-MYC. Moreover, tests of the efficacy of viscumTT in vivo showing reduction of tumor volume confirmed the high therapeutic potential as an anti-tumoral agent for osteosarcoma.