Highly reliable GIGA-sized synthetic human therapeutic antibody library construction.

Background: Monoclonal 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. Methods: The 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 ß-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. Results: We 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. Conclusion: The 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.

Ambient air particulates-bound metallic elements sources identifications during winter and summer at a Science Park.

The ambient air particulates pollutants of total suspended particulates (TSP) and PM2.5 were collected by using PS-1 and Wilbur PM2.5 sampler, simultaneously during the year of 2015-2017 at a photoelectric factory in Science Park of central Taiwan. And those of the ambient air atmospheric metallic elements (Cr, Mn, Ni, Cu, Zn, Pb) concentrations which attached on the TSP and PM2.5 were analyzed by using inductively coupled plasma optical emission spectrometer. In addition, identifying anthropogenic and natural pollutants sources were conducted by using the enrichment factor (EF) and principal component analysis (PCA) methods. The results indicated that the average TSP and PM2.5 concentrations were ranked highest in winter season, while summer season was ranked lowest during the year of 2015-2016. In addition, the average highest metallic element concentrations were occurred in winter season for both TSP and PM2.5 during the year of 2015-2016, while the average lowest metallic elements concentrations in TSP and PM2.5 were also occurred in winter season during the year of 2016-2017. Moreover, the EF analysis results showed that the metallic element Zn came from anthropogenic emission source. As for metallic element Mn, the results showed that metallic element Mn was mainly attributed to natural emission in this study. Finally, the PCA results showed that metallic elements Cr, Zn and Pb were the dominant emissions metallic elements in this study. As for PM2.5, the results showed that the metallic elements Cr, Cu and Pb were the dominant emissions metallic elements at this HPB sampling site.

Wet depositions of mercury during plum rain season in Taiwan.

The plum rain season in Taiwan is in May and June. The severest plum rain season over the last 21 years was in 2017. This study involves the collection of mercury wet depositions in the plum rain season of May-June in 2017. A DMA-80 (Direct Mercury Analyzer) was used to analyze the precipitated mercury concentrations and calculate the wet depositions of mercury in the plum rain season. The results indicate that the highest wet depositions of mercury in the aqueous phase were on 6/16, reaching 209.04 µg/m2 * day, while the lowest were on 5/15, at 0.18 µg/m2 * day. The mercury wet depositions in the particulate phase were highest on 6/17, when it exceeded 100 µg/kg, and lowest on particulate phase were occurred in 6/11, when it was 3.64 µg/m2 * day. The relationship between the wet depositions of mercury in the aqueous phase and rainfall was insignificant, while that between the wet depositions of mercury in the particulate phase and rainfall was significant. The wet depositions of mercury in this study were second highest (30.73 µg/m2 day) when compared with those in studies in the years 2007-2017. Although the rainfall in this study was only 564 mm H2O, high mercury concentrations obtained from the plum rain season result in the high wet depositions of mercury in Taichung, Taiwan.

Concentrations and analysis of health risks of ambient air metallic elements at Longjing site in central Taiwan.

The concentrations of particulates and metallic elements that were bound to total suspended particulates in ambient air at Long Cyuan Elementary School (LCYES), Lung Ching Elementary School (LCHES) and Long Shan Primary School (LSPS) sampling sites in the Longjing area were measured. Significant difference tests were conducted at LSPS, LCYES and LCHES sites. Finally, carcinogenic and non-carcinogenic risk values for LSPS, LCYES and LCHES sites in the Longjing district were evaluated. The results show that the most average particulate and metallic element concentrations were highest in October, November, January, February, March, April, August, and September The average particulate and metallic element concentrations at LCHES were higher than at the other sampling sites. The Concentration Scatter Diagrams reveal the absence of significant variation among the LSPS, LCYES and LCHES sampling sites in the Longjing district. Therefore, these sampling sites are inferred to have similar emission sources. The children and adults inhalation carcinogenic risks which referenced US EPA method were all within acceptable ranges. Non-carcinogenic risks revealed that all metallic elements considered herein were harmless to human health.

Review of total suspended particles (TSP) and PM2.5 concentration variations in Asia during the years of 1998-2015.

In Asian countries such as China, Malaysia, Pakistan, India, Taiwan, Korea, Japan and Hong Kong, ambient air total suspended particulates and PM2.5 concentration data were collected and discussed during the years of 1998-2015 in this study. The aim of the present study was to (1) investigate and collect ambient air total suspended particulates (TSP) and PM2.5 concentrations for Asian countries during the past two decades. (2) Discuss, analyze and compare those particulates (TSP and PM2.5) annual concentration distribution trends among those Asian countries during the past two decades. (3) Test the mean concentration differences in TSP and PM2.5 among the Asian countries during the past decades. The results indicated that the mean TSP concentration order was shown as China > Malaysia > Pakistan > India > Taiwan > Korea > Japan. In addition, the mean PM2.5 concentration order was shown as Vietnam > India > China > Hong Kong > Mongolia > Korea > Taiwan > Japan and the average percentages of PM2.5 concentrations for Taiwan, China, Japan, Korea, Hong Kong, Mongolia and Other (India and Vietnam) were 8, 21, 6, 8, 14, 13 and 30%, respectively, during the past two decades. Moreover, t test results revealed that there were significant mean TSP and PM2.5 concentration differences for either China or India to any of the countries such as Taiwan, Korea and Japan in Asia during the past two decades for this study. Noteworthy, China and India are both occupied more than 60% of the TSP and PM2.5 particulates concentrations out of all the Asia countries. As for Taiwan, the average PM2.5 concentration displayed increasing trend in the years of 1998-1999. However, it showed decreasing trend in the years of 2000-2010. As for Korea, the average PM2.5 concentrations showed decreasing trend during the years of 2001-2013. Finally, the average PM2.5 concentrations for Mongolia displayed increasing trend in the years of 2004-2013.

Seasonal variations and sources study by way of back trajectories and ANOVA for ambient air pollutants (particulates and metallic elements) within a mixed area at Longjing, central Taiwan: 1-year observation.

This study measured the concentrations of particulates and metallic elements in ambient air by using PS-1 sampler (TSP) at Longjing area. And this study focuses on the collection of ambient air particulates, metallic elements, particulate-bound mercury Hg(p), concentrations. In addition, the sources of ambient pollutants by way of back trajectory analysis are found. Moreover, test mean concentration variance differences for metallic elements (PM, Hg(p), Mn, Fe, Zn, Cr, Cu, and Pb) among the four seasons (spring, summer, autumn and winter) through ANOVA are calculated. The result indicates that the average highest particulate concentration occurred in winter season, and the order was winter > spring > autumn > summer, and the mostly highest average metallic element (Mn, Fe, Zn, Cr, Cu, Pb) concentrations occurred in autumn. Moreover, the mostly average lowest metallic element concentrations occurred in summer. In addition, the above results of backward trajectories that the major particulate pollutants parcel mainly come from northeastern Taiwan. Moreover, when comparing the results of the first half year to that of the second half year, the they indicated that all metallic elements displayed significant differences in concentrations except those of Hg(p), Mn, Fe, Zn. Finally, metallic element Hg(p) is the only one which showed no significant concentration difference from either seasonal variations or half-year observations.

PM2.5 particulates and metallic elements (Ni, Cu, Zn, Cd and Pb) study in a mixed area of summer season in Shalu, Taiwan.

PM2.5 has become an important environmental issue in Taiwan during the past few years. Moreover, electricity increased significantly during the summertime and TTPP generated by coal burning base is the main electricity provider in central Taiwan. Therefore, summer season has become the main research target in this study. The ambient air concentrations of particulate matter PM2.5 and PM10 collected by using VAPS at a mixed characteristic sampling site were studied in central Taiwan. The results indicated that the average daytime PM2.5 and PM10 particulate concentrations were occurred in May and they were 44.75 and 57.77 µg/m3 in this study. The results also indicated that the average nighttime PM2.5 and PM10 particulate concentrations were occurred in June and they were 38.19 and 45.79 µg/m3 in this study. The average PM2.5/PM10 ratios were 0.7 for daytime, nighttime and 24-h sampling periods in the summer for this study. This value was ranked as the lowest ratios when compared to the other seasons in previous study. Noteworthy, the results further indicated that the metallic element Pb has the mean highest concentrations for 24-h, daytime and nighttime sampling periods when compared to those of the other metallic elements (Ni, Cu, Zn and Cd). The average mean highest metallic Pb concentrations in PM10 were 110.7, 203.0 and 207.2 ng/m3 for 24-h, daytime and nighttime sampling periods in this study. And there were 59.53, 105.2 and 106.6 ng/m3 for Pb in PM2.5 for 24-h, daytime and nighttime sampling periods, respectively. Moreover, the results further indicated that mean metallic element Pb concentrations on PM2.5 and PM10 were all higher than those of the other elements for 24 h, day and nighttime.

Annual ambient atmospheric mercury speciation measurement from Longjing, a rural site in Taiwan.

The main purpose of this study was to monitor ambient air particulates and mercury species [RGM, Hg(p), GEM and total mercury] concentrations and dry depositions over rural area at Longjing in central Taiwan during October 2014 to September 2015. In addition, passive air sampler and knife-edge surrogate surface samplers were used to collect the ambient air mercury species concentrations and dry depositions, respectively, in this study. Moreover, direct mercury analyzer was directly used to detect the mercury Hg(p) and RGM concentrations. The result indicated that: (1) The average highest RGM, Hg(p), GEM and total mercury concentrations, and dry depositions were observed in January, prevailing dust storm occurred in winter season was the possible major reason responsible for the above findings. (2) The highest average RGM, Hg(p), GEM and total mercury concentrations, dry depositions and velocities were occurred in winter. This is because that China is the largest atmospheric mercury (Hg) emitter in the world. Its Hg emissions and environmental impacts need to be evaluated. (3) The results indicated that the total mercury ratios of Kaohsiung to that of this study were 5.61. This is because that Kaohsiung has the largest industry density (~60 %) in Taiwan. (4) the USA showed average lower mercury species concentrations when compared to those of the other world countries. The average ratios of China/USA values were 89, 76 and 160 for total mercury, RGM and Hg(p), respectively, during the years of 2000-2012.

Continuous microfluidic assortment of interactive ligands (CMAIL).

Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 10(5) CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 10(9) individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

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.