APOBEC3-related mutations in the spike protein-encoding region facilitate SARS-CoV-2 evolution.

SARS-CoV-2 evolves gradually to cause COVID-19 epidemic. One of driving forces of SARS-CoV-2 evolution might be activation of apolipoprotein B mRNA editing catalytic subunit-like protein 3 (APOBEC3) by inflammatory factors. Here, we aimed to elucidate the effect of the APOBEC3-related viral mutations on the infectivity and immune evasion of SARS-CoV-2. The APOBEC3-related C > U mutations ranked as the second most common mutation types in the SARS-CoV-2 genome. mRNA expression of APOBEC3A (A3A), APOBEC3B (A3B), and APOBEC3G (A3G) in peripheral blood cells increased with disease severity. A3B, a critical member of the APOBEC3 family, was significantly upregulated in both severe and moderate COVID-19 patients and positively associated with neutrophil proportion and COVID-19 severity. We identified USP18 protein, a key molecule centralizing the protein-protein interaction network of key APOBEC3 proteins. Furthermore, mRNA expression of USP18 was significantly correlated to ACE2 and TMPRSS2 expression in the tissue of upper airways. Knockdown of USP18 mRNA significantly decreased A3B expression. Ectopic expression of A3B gene increased SARS-CoV-2 infectivity. C > U mutations at S371F, S373L, and S375F significantly conferred with the immune escape of SARS-CoV-2. Thus, APOBEC3, whose expression are upregulated by inflammatory factors, might promote SARS-CoV-2 evolution and spread via upregulating USP18 level and facilitating the immune escape. A3B and USP18 might be therapeutic targets for interfering with SARS-CoV-2 evolution.

Tumor Microenvironment Composition and Related Therapy in Hepatocellular Carcinoma.

Globally, primary liver cancer is the third leading cause of cancer death, and hepatocellular carcinoma (HCC) accounts for 75%-95%. The tumor microenvironment (TME), composed of the extracellular matrix, helper cells, immune cells, cytokines, chemokines, and growth factors, promotes the immune escape, invasion, and metastasis of HCC. Tumor metastasis and postoperative recurrence are the main threats to the long-term prognosis of HCC. TME-related therapies are increasingly recognized as effective treatments. Molecular-targeted therapy, immunotherapy, and their combined therapy are the main approaches. Immunotherapy, represented by immune checkpoint inhibitors (ICIs), and targeted therapy, highlighted by tyrosine kinase inhibitors (TKIs), have greatly improved the prognosis of HCC. This review focuses on the TME compositions and emerging therapeutic approaches to TME in HCC.

A dataset on 24-h electrocardiograph, sleep and metabolic function of male type 2 diabetes mellitus.

This dataset provides a collection of 24 h electrocardiograph (ECG) signals, ECG analysis results based on circadian rhythm and R-peak detection, results of sleep quality assessment and clinical indicators of metabolic function acquired from 60 male type 2 diabetes mellitus (T2DM) inpatients. Upon admission, a fasting blood draw and urinary sample were obtained the next morning for routine glucose, lipid and renal panels. Subjects were also involved in investigation for diabetic complications. On the second day of hospitalization, subjects were monitored in hospital for 24-h ECG starting at 10 pm. Subjective sleep quality was assessed by Pittsburgh Sleep Quality Index and a brief sleep log was used to record sleep duration for the studied night. Objective sleep quality and sleep staging were assessed by cardiopulmonary coupling analysis. This dataset could be utilized to conduct conjoint research on the relationships among sleep, metabolic function, and function of cardiovascular system and autonomic nervous system derived from ECG analysis in T2DM, and further investigate the information in ECG signals based on circadian rhythm and physiological status, providing new insights into long term physiological signal processing.

Heart rate variability in different sleep stages is associated with metabolic function and glycemic control in type 2 diabetes mellitus.

Introduction: Autonomic nervous system (ANS) plays an important role in the exchange of metabolic information between organs and regulation on peripheral metabolism with obvious circadian rhythm in a healthy state. Sleep, a vital brain phenomenon, significantly affects both ANS and metabolic function. Objectives: This study investigated the relationships among sleep, ANS and metabolic function in type 2 diabetes mellitus (T2DM), to support the evaluation of ANS function through heart rate variability (HRV) metrics, and the determination of the correlated underlying autonomic pathways, and help optimize the early prevention, post-diagnosis and management of T2DM and its complications. Materials and methods: A total of 64 volunteered inpatients with T2DM took part in this study. 24-h electrocardiogram (ECG), clinical indicators of metabolic function, sleep quality and sleep staging results of T2DM patients were monitored. Results: The associations between sleep quality, 24-h/awake/sleep/sleep staging HRV and clinical indicators of metabolic function were analyzed. Significant correlations were found between sleep quality and metabolic function (|r| = 0.386 ± 0.062, p < 0.05); HRV derived ANS function showed strengthened correlations with metabolic function during sleep period (|r| = 0.474 ± 0.100, p < 0.05); HRV metrics during sleep stages coupled more tightly with clinical indicators of metabolic function [in unstable sleep: |r| = 0.453 ± 0.095, p < 0.05; in stable sleep: |r| = 0.463 ± 0.100, p < 0.05; in rapid eye movement (REM) sleep: |r| = 0.453 ± 0.082, p < 0.05], and showed significant associations with glycemic control in non-linear analysis [fasting blood glucose within 24 h of admission (admission FBG), |r| = 0.420 ± 0.064, p < 0.05; glycated hemoglobin (HbA1c), |r| = 0.417 ± 0.016, p < 0.05]. Conclusions: HRV metrics during sleep period play more distinct role than during awake period in investigating ANS dysfunction and metabolism in T2DM patients, and sleep rhythm based HRV analysis should perform better in ANS and metabolic function assessment, especially for glycemic control in non-linear analysis among T2DM patients.

The mortalities of female-specific cancers in China and other countries with distinct socioeconomic statuses: A longitudinal study.

INTRODUCTION: Female-specific cancers seriously affect physical and psychological health of women worldwide. OBJECTIVES: We aimed to elucidate trends in the age-standardized mortality rates (ASMRs) of breast cancer, cervical cancer, uterine cancer, and ovarian cancer in female populations with different socioeconomic statuses in China and in countries with different Human Development Index (HDI). METHODS: A longitudinal study was performed using the data of cancer death in China and other 39 countries. The mortality rates were standardized with the Segi's world population. Trends in the mortalities were exhibited by estimated annual percentage change (EAPC). Pearson correlation was used to assess the association between EAPC and HDI. RESULTS: In mainland China, female breast cancer, cervical cancer, uterine cancer, and ovarian cancer accounted for 6.60 %, 4.21 %, 2.50 %, and 2.02 % of cancer death (n = 1,314,040) in women with 1,220,251,032 person-years, respectively. The ASMRs of cervical cancer (EAPC = 3.87 %, P < 0.001) and ovarian cancer (EAPC = 1.81 %, P < 0.001) increased, that of female breast cancer unchanged, whereas that of uterine cancer was extremely higher and rapidly decreased (EAPC =  - 7.65 %, P < 0.001), during 2004-2019. The ASMRs of female breast and ovarian cancers were higher in urban and developed regions than in rural and undeveloped regions, in contrast to cervical and uterine cancers. The ASMRs of female breast and ovarian cancers were lower in China than in other countries, in contrast to uterine cancer. The ASMR of cervical cancer decreased, that of uterine cancer increased, in other countries during 2004-2017. EAPCs for the ASMRs of breast and ovarian cancers were inversely correlated to HDI. CONCLUSION: The ASMRs of cervical and ovarian cancers increased, in contrast to uterine cancer, in China during socioeconomic transition. Trends in the ASMRs of breast and ovarian cancers were inversely associated with HDI. These data help control female-specific cancers.

Tumor-Targeting Ability of Novel Anti-Prostate-Specific Membrane Antigen Antibodies.

Patients with prostate-specific membrane antigen (PSMA)-positive tumors can benefit from PSMA-targeted therapy; thus, we have constructed a phage-displayed synthetic antibody library for the production of novel PSMA antibodies with superior PSMA-targeting ability, favoring clinical management. The binding affinities of anti-PSMA antibodies were verified by an enzyme-linked immunosorbent assay (ELISA). Several in vitro and in vivo experiments, including cellular uptake, internalization, and cytotoxicity studies, micro single photon emission computed tomography (microSPECT)/CT, and biodistribution studies, were performed to select the most promising antibody among six different antibodies. The results showed the target affinities of our antibodies in the ELISA assays (7A, 8C, 8E, and 11A) were comparable to the existing antibodies (J591). The half-maximal effective concentrations of 7A, 8C, 8E, 11A, and J591 were 2.95, 6.64, 5.50, 2.08, and 4.79, respectively. The radiochemical yield of 111In-labeled antibodies ranged from 30% to 50% with high radiochemical purity (>90%). In the cellular uptake studies, the accumulated radioactivity of 111In-J591, 111In-7A, and 111In-11A increased over time. The internalized percentage of 111In-11A was the highest (32.14% ± 2.06%) at 48 h after incubation, whereas that of 111In-J591 peaked at 22.43% ± 4.38% at 24 h and dropped to 13.52% ± 3.03% at 48 h postincubation. Twenty-four hours after injection, radioactivity accumulation appeared in the LNCaP xenografts of the mice injected with 111In-11A, 111In-8E, 111In-7A, and 111In-J591 but not in the xenografts of the 111In-8C-injected group. Marked liver uptake was noticed in all groups except the 111In-11A-injected group. Moreover, the killing effect of 177Lu-11A was superior to that of 177Lu-J591 at low concentrations. In conclusion, we successfully demonstrated that 11A IgG owned the most optimal biological characteristics among several new anti-PSMA antibodies and it can be an excellent PSMA-targeting component for the clinical use.

Eradicating mesothelin-positive human gastric and pancreatic tumors in xenograft models with optimized anti-mesothelin antibody-drug conjugates from synthetic antibody libraries.

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody-drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen.

Structure-based Development of Human Interleukin-1ß-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP.

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.

Preparation and characterization of antibody-drug conjugates acting on HER2-positive cancer cells.

Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate.

A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed synthetic antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes.

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10-15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.

Circulating galectin-3 on admission and prognosis in acute heart failure patients: a meta-analysis.

Changes of serum galectin-3 have been associated with the pathogenesis of many cardiovascular diseases. The aim of the study was to evaluate the prognostic role of serum galectin-3 in patients with acute heart failure (AHF) in a meta-analysis. Follow-up studies evaluating the association between serum galectin-3 on admission and clinical outcomes in AHF patients were identified via search of PubMed and Embase databases. A random effects or a fixed effects model was applied to pool the results depending on the heterogeneity. Subgroup analysis was used to evaluate the influences of study characteristics on the outcomes. Overall, 7057 AHF patients from eighteen follow-up studies were included. Higher serum galectin-3 was associated with higher risks of all-cause mortality (adjusted risk ratio [RR], 1.58; p < 0.001), mortality/HF rehospitalization (RR, 1.68; p < 0.001), and cardiovascular mortality (RR, 1.29; p = 0.04), but not HF rehospitalization (RR, 1.24; p = 0.25) in AHF patients. Subgroup analyses showed that study characteristics including study design, sample size, age, gender, left ventricular ejection fraction, galectin-3 variable type, follow-up duration, and adjustment of type B natriuretic peptide did not significantly impact the results. Significant heterogeneities were detected for the outcomes of all-cause mortality and mortality/HF rehospitalization. However, trim-and-fill analyses by including the imputed studies to generate symmetrical funnel plots showed similar significant meta-analysis results. These results suggested that higher serum galectin-3 may be associated with poor prognosis in AHF patients. Further studies are needed to determine the mechanisms underlying the potential prognostic role of galectin-3 in AHF.

Development of theranostic active-targeting boron-containing gold nanoparticles for boron neutron capture therapy (BNCT).

Successful boron neutron capture therapy (BNCT) requires sufficient and specific delivery of boron atoms to malignant cells. Gold nanoparticles (AuNPs) have been used as a useful delivery system for selectively releasing cytotoxic payloads in the tumor. However, studies demonstrating the in vivo distribution or pharmacokinetics of boron-containing AuNPs via noninvasive imaging are lacking. This study aims to develop theranostic AuNP-boron cage assemblies (B-AuNPs) and evaluate its feasibility for BNCT. The commercial citrate-coated AuNPs were subjected to PEGylation, azide addition, and carborane modification on the surface. To further arm the AuNPs, we conjugated anti-HER2 antibody (61 IgG) with boron-containing PEGylated AuNPs to form 61-B-AuNPs. The diameter and radiolabeling efficiency of boron-containing AuNPs were determined by dynamic light scattering (DLS) and radio thin-layer chromatography (radio TLC), respectively. Noninvasive single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging was performed to determine the pharmacokinetics of radioiodinated AuNPs in N87 gastric cancer xenografts, and the content of boron in tumor and muscle was assessed by inductively coupled plasma mass spectrometry (ICP-MS). After the 3-step modification, the diameter of B-AuNPs increased by ˜25 nm, and antibody conjugation did not affect the diameter of AuNPs. Radioactive iodine (I-123) was introduced in AuNPs by Click chemistry under copper catalysis. The radiolabeling efficiency of 123I-B-AuNPs and 123I-61-B-AuNPs was approximately 60 ± 5%. After purification, the radiochemical purity (RCP) of these NPs was greater than 90%. MicroSPECT/CT imaging showed that the tumor-to-muscle (T/M) ratio of 123I-B-AuNP-injected mice reached 1.91 ± 0.17 at 12 h post-injection, while that of 123I-61-B-AuNP-injected mice was 12.02 ± 0.94. However, the increased uptake of AuNPs by the thyroid was observed at 36 h after the administration of 123I-61-B-AuNPs, indicating antibody-mediated phagocytosis. The T/M ratio, assessed by ICP-MS, of B-AuNP- and 61-B-AuNP-injected mice was 4.91 ± 2.75 and 41.05 ± 11.15, respectively. We successfully developed detectable HER2-targeting boron-containing AuNPs with high RCP and an acceptable yield. Noninvasive imaging could be a valuable tool for the noninvasive determination of the pharmacokinetics of AuNPs and measurement of boron concentration in the tumor.

Efficacy and Safety of Antiplatelet Therapy Plus Xa Factor Inhibitors in Patients with Coronary Heart Disease: A Meta-Analysis.

BACKGROUND The aim of this study was to systematically evaluate the effect of oral Xa inhibitors plus antiplatelet therapy in the treatment of coronary artery disease. MATERIAL AND METHODS All randomized controlled trials (RCTs) about antiplatelet therapy plus Xa factor inhibitors for coronary artery disease from database inception to January 2019 were searched for and collected from PubMed, Embase, and the Cochrane Library. Two reviewers extracted and analyzed the data independently. Additionally, RevMan 5.0 software was applied for meta-analysis. RESULTS Seven RCTs with 50 044 patients were included. The meta-analysis results showed that treatment with antiplatelet therapy plus Xa factor inhibitors in patients with coronary artery disease could significantly reduce the risk of ischemic events (P<0.00001). Besides, risk of all-cause mortality (P=0.003), myocardial infarction (P=0.02) and ischemic stroke (P<0.0001) were also significantly reduced. However, risk of massive hemorrhage after TIMI (P<0.00001), minor hemorrhage after TIMI (P<0.00001), and intracranial hemorrhage (P=0.006) were significantly increased, respectively. Xa inhibition drugs also intended to increase risk of fatal bleeding, but there was no significant difference (P=0.08). CONCLUSIONS Antiplatelet therapy plus Xa factor inhibitors in patients with coronary artery disease was effective, which could reduce the risk of ischemic composite endpoints, all-cause mortality, myocardial infarction, and ischemic stroke. However, it could significantly increase risk of bleeding in terms of safety.

Effective binding to protein antigens by antibodies from antibody libraries designed with enhanced protein recognition propensities.

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models.

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.

Noninvasive assessment of characteristics of novel anti-HER2 antibodies by molecular imaging in a human gastric cancer xenograft-bearing mouse model.

Human epidermal growth factor receptor 2 (HER2) overexpression occurs in various types of cancers. Regarding the anti-HER2 targeted therapies showed superior treatment outcomes in several (pre)clinical studies, we used multimodality image to rapidly select novel HER2-targeting antibodies for further therapeutics development. The four anti-HER2 antibodies (H32 IgG, 75 IgG, 61 IgG, and trastuzumab) labeled with either In-111 or a DyLight680 fluorescent dye were applied to perform cellular uptake, endocytosis, optical/microSPECT/CT imaging and biodistribution studies. In vitro and in vivo relative effectiveness of these antibodies were also compared in an N87 gastric cancer xenograft model. The internalized radioactivity of [111In]61 IgG in N87 cells increased from 33% at 12 hr to 56% at 48 hr after incubation, while the majority of other antibodies stayed on the cell membranes. Among these antibodies, 61 IgG showed the highest accumulation in tumors with the tumor-to-muscle ratio (T/M) of 131 ± 61.4 and 19.13 ± 3.42 conducted by IVIS and microSPECT/CT, respectively. We demonstrated that multimodality imaging is a reliable approach for selecting potential antibodies and found that 61 IgG manifested significant tumor accumulation with elevated internalization rate thus could be a suitable candidate for further development of new HER2-targeted therapies.

High throughput discovery of influenza virus neutralizing antibodies from phage-displayed synthetic antibody libraries.

Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural antibody repertoires. With continuing advancement in designing and constructing synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.

Effects of gripping volume in the mechanical strengths of orthodontic mini-implant.

The objective of study was to investigate the correlation between the mechanical strengths [insertion torque (IT); resonance frequency (RF); and horizontal pullout strength (HPS)] and gripping volume (GV) of mini-implants. Thirty mini-implants of three types (Type A: 2 mm × 10 mm, cylindrical, titanium alloy; Type B: 2 mm × 10 mm, tapered, stainless steel; and Type C: 2 mm × 11 mm, cylindrical, titanium alloy) were inserted 7 mm into artificial bones. One-way analysis of variance and Spearman's test were applied to assess intergroup comparisons and intragroup correlations. The null hypothesis was that no statistically significant correlations exist between the GV and mechanical strengths (IT, RF, and HPS). In the IT test, Type C (14.2 Ncm) had significantly (p=0.016) greater values than did Type A (12.4 Ncm). In the RF analysis, no significant difference was observed among the three types of mini-implants. In the HPS test, Type C (388.9 Ncm) was significantly larger than both Type B (294.5 Ncm) and Type A (286 Ncm). In the GV measurement, Type C (14.4 mm3) was significantly larger than Type B (11.4 mm3) and Type A (9.2 mm3). Type A and Type B exhibited no significant correlations among the tests. Therefore, the null hypothesis was accepted. Although no significant correlation was noted between the GV and mechanical strengths (IT, RF, and HPS), we observed a trend that the mechanical strengths (IT, RF, and HPS) of the mini-implants corresponded to the order and values of GV (Type C > Type B > Type A).

Effect of Anchor Length on the Pullout Strength of Palatal Mini Implants.

PURPOSE: The aim of this study was to evaluate the pullout strength of 3 different orthodontic mini implants. MATERIALS AND METHODS: Twenty-seven mini implants (diameter: 2 mm, length: 7 mm) were implanted into artificial bone (Sawbones; Pacific Research Laboratories Inc.) at depths of 3, 4, and 5 mm. The insertion torque (IT), resonance frequency (RF), pullout strength (PS), and anchor length (AL) were measured. One-way analysis of variance with Tukey honest significant difference (HSD) postcomparison were used to detect intergroup differences. The null hypothesis was that IT, RF, and PS would significantly correlate in the same brand. RESULTS: In the implantation depths (ID) (5 and 4 mm), IT of Types C (16.67 and 14.33 N·cm) and Type B (14 and 13.33 N·cm) were significantly higher than Type A (10.33 and 9.33 N·cm). Type A had a largest AL and PS at the IDs (5 and 4 mm). In the IDs (3 mm), PS was no different. Type C had no correlation among the RF, IT, and PS. Therefore, null hypothesis was rejected. CONCLUSION: AL exerted crucial effects on the PS of the mini implants.