Highly reliable GIGA-sized synthetic human therapeutic antibody library construction

BackgroundMonoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library’s potential for biomedical applications.MethodsThe library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to β-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays.ResultsWe have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation.ConclusionThe promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies

Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2

Dengue virus (DENV) infection remains a serious health threat despite the availability of supportive care in modern medicine. Monoclonal antibodies (mAbs) of DENV would be powerful research tools for antiviral development, diagnosis and pathological investigations. Here we described generation and characterization of seventeen mAbs with high reactivity for E protein of DENV. Four of these mAbs showed high neutralizing activity against DENV-2 infection in mice. The monoclonal antibody mAb DB32-6 showed the strongest neutralizing activity against diverse DENV-2 and protected DENV-2-infected mice against mortality in therapeutic models. We identified neutralizing epitopes of DENV located at residues K310 and E311 of viral envelope protein domain III (E-DIII) through the combination of biological and molecular strategies. Comparing the strong neutralizing activity of mAbs targeting A-strand with mAbs targeting lateral ridge, we found that epitopes located in A-strand induced stronger neutralizing activity than those located on the lateral ridge. DB32-6 humanized version was successfully developed. Humanized DB32-6 variant retained neutralizing activity and prevented DENV infection. Understanding the epitope-based antibody-mediated neutralization is crucial to controlling dengue infection. Additionally, this study also introduces a novel humanized mAb as a candidate for therapy of dengue patients

Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level

Graduates’ and employers’ perceptions on competencies obtained from an undergraduate dental curriculum

The effectiveness of a dental curriculum as reflected by the competencies of the graduates is paramount in ensuring its relevance in the rapidly evolving field of dentistry. This work aimed to assess the competencies of dental graduates of the Faculty of Dentistry, Universiti Kebangsaan Malaysia (UKM) as perceived by the graduates and their employers on the basis of the core competencies listed in the undergraduate dental curriculum. A self-administered questionnaire consisting of 43 questions based on eight competency domains were sent to all UKM dental graduates of years 2012 to 2015 and to senior dental officers who represented their employers. The eight domains assessed were as follows: 1) gathering information at chair-side, 2) diagnosis, 3) treatment planning, 4) treatment and prevention, 5) community-based, 6) management and administrative, 7) communication and 8) personal management and professional development. a total of 132 graduates (75%) and 18 employers (55%) responded. Only domain E on community-based skills was collectively rated ‘satisfactory’ while the rest of the seven domains had an ‘excellent’ rating by all respondents. With regard to individual skill, basic life support was rated very low for both groups (38.6% graduates and 23.5% employers). A strong association was found between the scores given by the graduates and their employers (p= 0.00). Generally, no difference was found between the scores of graduates from different years. The findings allow an evaluation of the curriculum in a myriad of angles. Although the graduates reported a good level of competency in most domains, the employers were reserved on the community-based skills. Poor competency in the key skill of basic life support in medical emergencies was highlighted and must be addressed in the curriculum or during training at the workplace

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy

Robot-based intervention may reduce delay in the production of intransitive gestures in Chinese-speaking preschoolers with autism spectrum disorder

Abstract Background Past studies have shown that robot-based intervention was effective in improving gestural use in children with autism spectrum disorders (ASD). The present study examined whether children with ASD could catch up to the level of gestural production found in age-matched children with typical development and whether they showed an increase in verbal imitation after the completion of robot-based training. We also explored the cognitive and motor skills associated with gestural learning. Methods Children with ASD were randomly assigned to two groups. Four- to 6-year-old children with ASD in the intervention group (N = 15) received four 30-min robot-based gestural training sessions. In each session, a social robot, NAO, narrated five stories and gestured (e.g., both hands clapping for an awesome expression). Children with ASD were told to imitate the gestures during training. Age-matched children with ASD in the wait-list control group (N = 15) and age-matched children with typical development (N = 15) received the gestural training after the completion of research. Standardized pretests and posttests (both immediate and delayed) were administered to assess the accuracy and appropriateness of gestural production in both training and novel stories. Children’s language and communication abilities, gestural recognition skills, fine motor proficiencies, and attention skills were also examined. Results Children with ASD in the intervention condition were more likely to produce accurate or appropriate intransitive gestures in training and novel stories than those in the wait-list control. The positive learning outcomes were maintained in the delayed posttests. The level of gestural production accuracy in children with ASD in the delayed posttest of novel stories was comparable to that in children with typical development, suggesting that children with ASD could catch up to the level of gestural production found in children with typical development. Children with ASD in the intervention condition were also more likely to produce verbal markers while gesturing than those in the wait-list control. Gestural recognition skills were found to significantly predict the learning of gestural production accuracy in the children with ASD, with such relation partially mediated via spontaneous imitation. Conclusions Robot-based intervention may reduce the gestural delay in children with ASD in their early childhood

Psychometric Properties of the Internet Gaming Disorder Scale–Short-Form (IGDS9-SF): Systematic Review

BackgroundThe Internet Gaming Disorder Scale–Short-Form (IGDS9-SF) is among the best with regard to its psychometric properties. Therefore, clinical psychologists are likely guided to use the IGDS9-SF if they want to assess or screen the disordered gaming in their practice. However, the information, especially psychometric evidence, concerning the IGDS9-SF has not been fully examined and summarized. ObjectiveThis systematic review evaluated the psychometric properties of different language versions of the IGDS9-SF and assessed its methodological quality in order to improve the clinicians’ understanding of the IGDS9-SF and facilitate its use. MethodsSystematic literature searches were carried out using Embase, MEDLINE, PsycINFO, PubMed, ScienceDirect, Scopus, and Web of Science. The review included English-language studies of any research design that have reported at least one psychometric property of the IGDS9-SF, as defined by the COnsensus-based Standards for the selection of health status Measurement INstrument (COSMIN), and have aimed at testing the psychometric properties of the IGDS9-SF. ResultsIn total, 21 studies comprising 15 language versions of the IGDS9-SF were included. Overall, the IGDS9-SF showed adequate internal consistency (although some items did not have satisfactory item-total correlation [IT]), excellent criterion validity, and the ability to distinguish different subgroups with measurement invariance being supported across gender and age. In terms of factor structure, the IGDS9-SF was shown to have a unidimensional factor structure across all 21 studies. ConclusionsAlthough there is insufficient evidence regarding the responsiveness and properties of the IGDS9-SF using item response theory, the existing evidence supports its use in assessing disordered gaming among individuals

Identify potential drugs for cardiovascular diseases caused by stress-induced genes in vascular smooth muscle cells

Background Abnormal proliferation of vascular smooth muscle cells (VSMC) is a major cause of cardiovascular diseases (CVDs). Many studies suggest that vascular injury triggers VSMC dedifferentiation, which results in VSMC changes from a contractile to a synthetic phenotype; however, the underlying molecular mechanisms are still unclear. Methods In this study, we examined how VSMC responds under mechanical stress by using time-course microarray data. A three-phase study was proposed to investigate the stress-induced differentially expressed genes (DEGs) in VSMC. First, DEGs were identified by using the moderated t-statistics test. Second, more DEGs were inferred by using the Gaussian Graphical Model (GGM). Finally, the topological parameters-based method and cluster analysis approach were employed to predict the last batch of DEGs. To identify the potential drugs for vascular diseases involve VSMC proliferation, the drug-gene interaction database, Connectivity Map (cMap) was employed. Success of the predictions were determined using in-vitro data, i.e. MTT and clonogenic assay. Results Based on the differential expression calculation, at least 23 DEGs were found, and the findings were qualified by previous studies on VSMC. The results of gene set enrichment analysis indicated that the most often found enriched biological processes are cell-cycle-related processes. Furthermore, more stress-induced genes, well supported by literature, were found by applying graph theory to the gene association network (GAN). Finally, we showed that by processing the cMap input queries with a cluster algorithm, we achieved a substantial increase in the number of potential drugs with experimental IC50 measurements. With this novel approach, we have not only successfully identified the DEGs, but also improved the DEGs prediction by performing the topological and cluster analysis. Moreover, the findings are remarkably validated and in line with the literature. Furthermore, the cMap and DrugBank resources were used to identify potential drugs and targeted genes for vascular diseases involve VSMC proliferation. Our findings are supported by in-vitro experimental IC50, binding activity data and clinical trials. Conclusion This study provides a systematic strategy to discover potential drugs and target genes, by which we hope to shed light on the treatments of VSMC proliferation associated diseases