Publications by authors named "Keyang Xu"

58 Publications

Investigation of the Mechanism of Complement System in Diabetic Nephropathy via Bioinformatics Analysis.

J Diabetes Res 2021 24;2021:5546199. Epub 2021 May 24.

Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, 610072 Sichuan, China.

Objectives: Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD) throughout the world, and the identification of novel biomarkers via bioinformatics analysis could provide research foundation for future experimental verification and large-group cohort in DN models and patients.

Methods: GSE30528, GSE47183, and GSE104948 were downloaded from Gene Expression Omnibus (GEO) database to find differentially expressed genes (DEGs). The difference of gene expression between normal renal tissues and DN renal tissues was firstly screened by GEO2R. Then, the protein-protein interactions (PPIs) of DEGs were performed by STRING database, the result was integrated and visualized via applying Cytoscape software, and the hub genes in this PPI network were selected by MCODE and topological analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to determine the molecular mechanisms of DEGs involved in the progression of DN. Finally, the Nephroseq v5 online platform was used to explore the correlation between hub genes and clinical features of DN.

Results: There were 64 DEGs, and 32 hub genes were identified, enriched pathways of hub genes involved in several functions and expression pathways, such as complement binding, extracellular matrix structural constituent, complement cascade related pathways, and ECM proteoglycans. The correlation analysis and subgroup analysis of 7 complement cascade-related hub genes and the clinical characteristics of DN showed that C1QA, C1QB, C3, CFB, ITGB2, VSIG4, and CLU may participate in the development of DN.

Conclusions: We confirmed that the complement cascade-related hub genes may be the novel biomarkers for DN early diagnosis and targeted treatment.
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http://dx.doi.org/10.1155/2021/5546199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169258PMC
May 2021

Calicheamicin Antibody-Drug Conjugates with Improved Properties.

Mol Cancer Ther 2021 Jun 15;20(6):1112-1120. Epub 2021 Mar 15.

Genentech, Inc., South San Francisco, California.

Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States: Mylotarg (gemtuzumab ozogamicin) targeting CD33 for acute myeloid leukemia and Besponsa (inotuzumab ozogamicin) targeting CD22 for acute lymphocytic leukemia. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid-sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation, and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2 breast cancer) and hematologic malignancies (CD22 non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared with that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next-generation calicheamicin ADC.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0035DOI Listing
June 2021

Based on Network Pharmacology Tools to Investigate the Molecular Mechanism of Cordyceps sinensis on the Treatment of Diabetic Nephropathy.

J Diabetes Res 2021 5;2021:8891093. Epub 2021 Feb 5.

Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, 610072 Sichuan, China.

Background: Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus and is a major cause of end-stage kidney disease. Cordyceps sinensis (Cordyceps, Dong Chong Xia Cao) is a widely applied ingredient for treating patients with DN in China, while the molecular mechanisms remain unclear. This study is aimed at revealing the therapeutic mechanisms of Cordyceps in DN by undertaking a network pharmacology analysis.

Materials And Methods: In this study, active ingredients and associated target proteins of Cordyceps sinensis were obtained via Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and Swiss Target Prediction platform, then reconfirmed by using PubChem databases. The collection of DN-related target genes was based on DisGeNET and GeneCards databases. A DN-Cordyceps common target interaction network was carried out via the STRING database, and the results were integrated and visualized by utilizing Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to determine the molecular mechanisms and therapeutic effects of Cordyceps on the treatment of DN.

Results: Seven active ingredients were screened from Cordyceps, 293 putative target genes were identified, and 85 overlapping targets matched with DN were considered potential therapeutic targets, such as TNF, MAPK1, EGFR, ACE, and CASP3. The results of GO and KEGG analyses revealed that hub targets mainly participated in the AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, PI3K-Akt signaling pathway, and IL-17 signaling pathway. These targets were correlated with inflammatory response, apoptosis, oxidative stress, insulin resistance, and other biological processes.

Conclusions: Our study showed that Cordyceps is characterized as multicomponent, multitarget, and multichannel. Cordyceps may play a crucial role in the treatment of DN by targeting TNF, MAPK1, EGFR, ACE, and CASP3 signaling and involved in the inflammatory response, apoptosis, oxidative stress, and insulin resistance.
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http://dx.doi.org/10.1155/2021/8891093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884116PMC
February 2021

Systematic Variation of Pyrrolobenzodiazepine (PBD)-Dimer Payload Physicochemical Properties Impacts Efficacy and Tolerability of the Corresponding Antibody-Drug Conjugates.

J Med Chem 2020 09 18;63(17):9603-9622. Epub 2020 Aug 18.

Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States.

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of and experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00691DOI Listing
September 2020

Immunoaffinity LC-MS/MS is more suitable than ELISA to quantify a PEGylated molecule in cynomolgus monkey serum.

Bioanalysis 2020 Aug 31;12(15):1061-1069. Epub 2020 Jul 31.

Department of Bioanalytical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Polyethylene glycolylation (PEGylation) technology is a long-acting delivery platform used to increase the half-life of protein therapeutics. Quantitation of PEGylated anti-Factor D Fab (PEG-aFD) poses bioanalytical challenges. An ELISA was developed to determine total Fab concentration in cynomolgus monkey serum following intravitreal administration of PEG-aFD. However, assay characterization showed a low recovery of about 25% for free unconjugated Fab whereas recovery for PEG-conjugated Fab was within 80-120%. To overcome this challenge, an immunoaffinity liquid chromatography tandem mass spectrometry (IA LC-MS/MS) assay was developed, achieving recovery within 80-120% for both free and conjugated Fab. Immunoaffinity LC-MS/MS is more suitable than ELISA to accurately quantify the total protein concentration of PEG-aFD in cynomolgus monkey serum.
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http://dx.doi.org/10.4155/bio-2020-0097DOI Listing
August 2020

Automated, Generic Reagent and Ultratargeted 2D-LC-MS/MS Enabling Quantification of Biotherapeutics and Soluble Targets down to pg/mL Range in Serum.

Anal Chem 2020 07 18;92(13):9412-9420. Epub 2020 Jun 18.

Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States.

Mass spectrometry has recently emerged as a powerful analytical tool for the assessment of pharmacokinetics and biomarkers in drug development. Compared with ligand binding assays, a major advantage of mass spectrometry-based assays is that they are less dependent on high quality binding reagents, while a key limitation is the relatively lower sensitivity. To address the sensitivity issue, we have developed a generic reagent, ultratargeted two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method which combines commercially available protein A affinity capture, targeted analyte isolation by 2D-LC, and targeted detection by multiple reaction monitoring (MRM). A targeted-2D-with-dilution configuration was designed to automate 2D-LC-MS/MS. This method was systematically evaluated using an anti-CD22 monoclonal antibody spiked into monkey and human serum, where lower limits of quantification (LLOQ) of 0.78 and 1.56 ng/mL were achieved, respectively. This represents an over 100-fold improvement in assay sensitivity compared to the conventional LC-MS/MS method. The performance of the method was further confirmed by analyzing another monoclonal antibody, bevacizumab, as well as a soluble antigen, circulating PD-L1. The results indicate that our method enables quantification of antibody therapeutics and antigen biomarkers in both clinical and nonclinical samples in the pg/mL to low ng/mL range. Protein A affinity capture was employed as a universal sample preparation procedure applicable to both full-length antibody therapeutics and antibody-antigen complexes. This novel method is also fully automated and proven to be highly robust for routine bioanalysis in drug development.
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http://dx.doi.org/10.1021/acs.analchem.0c01910DOI Listing
July 2020

Role of identified noncoding RNA in erectile dysfunction.

Andrologia 2020 Aug 22;52(7):e13596. Epub 2020 May 22.

Hangzhou Xixi Hospital affiliated to Zhejiang Chinese Medical University, Hangzhou, China.

Erectile dysfunction (ED) is a common male sexual dysfunction and is closely related to many risk factors such as age, chronic diseases and mental disorder. Phosphodiesterase type 5 inhibitor (PDE5i) is recommended as the first-line medicine in therapy, but up to 35% of patients fail to this treatment. Unfortunately, the pathogenesis of ED is still poorly understood. Hence, it has reached the state that researchers should seek for new candidate biomarkers or therapeutic targets. Recent studies have reported that noncoding RNAs (ncRNAs) such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are involved in the pathogenesis process of ED, even in stem cell therapy. In this review, we aim to summarise the mechanisms and functions of identified ncRNAs that are associated with ED.
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http://dx.doi.org/10.1111/and.13596DOI Listing
August 2020

Adipose-tissue derived porcine mesenchymal stem cells efficiently ameliorate CCl-induced acute liver failure in mice.

Cytotechnology 2020 Jun 25;72(3):327-341. Epub 2020 Apr 25.

Storr Liver Centre, Westmead Institute for Medical Research, The University of Sydney and Westmead Hospital, Westmead, NSW, 2145, Australia.

Adipose tissue derived mesenchymal stem cells (ADMSCs) may be an attractive therapeutic source for acute liver injury because of their high accessibility and non-invasiveness. Here, we investigated the therapeutic potentials of porcine ADMSCs for acute liver failure (ALF). The morphology, differentiation potential, expression patterns of cell surface markers and liver-specific genes were compared between the ADMSCs derived from the pigs with or without ALF. For therapeutic studies, the expanded porcine ADMSCs from either ALF pig (ALF-ADMSCs) or healthy control pig (Nor-ADMSCs) of passage 3 were transplanted into CCl-induced ALF mice, and the liver histology and functional tests were performed at days 1, 7, 14, and 21 after cell transplantation. ALF-ADMSCs expressed higher mRNA level of hepatic growth factor (HGF) than the Nor-ADMSCs. Both ALF-ADMSCs and Nor-ADMSCs improved liver histology, functions, and mouse survival rate. Higher level of porcine hepatocyte-specific genes was seen in the livers of ALF-ADMSCs transplanted mice as compared to the Nor-ADMSCs transplanted mice. In particular, ALF-ADMSCs transplanted mice expressed significantly higher level of albumin and cytokeratin 18 in the liver tissues as compared to the Nor-ADMSCs transplanted mice. ALF-ADMSCs might be superior to Nor-ADMSCs in the treatment of ALF as the former possesses stronger hepatic differentiation potential.
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http://dx.doi.org/10.1007/s10616-020-00370-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225243PMC
June 2020

RNA 6-methyladenosine: a promising molecular target in metabolic diseases.

Cell Biosci 2020 21;10:19. Epub 2020 Feb 21.

4Hangzhou Xixi Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, 310023 Zhejiang China.

6-methyladenosine is a prevalent and abundant transcriptome modification, and its methylation regulates the various aspects of RNAs, including transcription, translation, processing and metabolism. The methylation of 6-methyladenosine is highly associated with numerous cellular processes, which plays important roles in the development of physiological process and diseases. The high prevalence of metabolic diseases poses a serious threat to human health, but its pathological mechanisms remain poorly understood. Recent studies have reported that the progression of metabolic diseases is closely related to the expression of RNA 6-methyladenosine modification. In this review, we aim to summarize the biological and clinical significance of RNA 6-methyladenosine modification in metabolic diseases, including obesity, type 2 diabetes, non-alcoholic fatty liver disease, hypertension, cardiovascular diseases, osteoporosis and immune-related metabolic diseases.
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http://dx.doi.org/10.1186/s13578-020-00385-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035649PMC
February 2020

Protein Biomarker Quantification by Immunoaffinity Liquid Chromatography-Tandem Mass Spectrometry: Current State and Future Vision.

Clin Chem 2020 02;66(2):282-301

Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN.

Immunoaffinity-mass spectrometry (IA-MS) is an emerging analytical genre with several advantages for profiling and determination of protein biomarkers. Because IA-MS combines affinity capture, analogous to ligand binding assays (LBAs), with mass spectrometry (MS) detection, this platform is often described using the term hybrid methods. The purpose of this report is to provide an overview of the principles of IA-MS and to demonstrate, through application, the unique power and potential of this technology. By combining target immunoaffinity enrichment with the use of stable isotope-labeled internal standards and MS detection, IA-MS achieves high sensitivity while providing unparalleled specificity for the quantification of protein biomarkers in fluids and tissues. In recent years, significant uptake of IA-MS has occurred in the pharmaceutical industry, particularly in the early stages of clinical development, enabling biomarker measurement previously considered unattainable. By comparison, IA-MS adoption by CLIA laboratories has occurred more slowly. Current barriers to IA-MS use and opportunities for expanded adoption are discussed. The path forward involves identifying applications for which IA-MS is the best option compared with LBA or MS technologies alone. IA-MS will continue to benefit from advances in reagent generation, more sensitive and higher throughput MS technologies, and continued growth in use by the broader analytical community. Collectively, the pursuit of these opportunities will secure expanded long-term use of IA-MS for clinical applications.
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http://dx.doi.org/10.1093/clinchem/hvz022DOI Listing
February 2020

Improved translation of stability for conjugated antibodies using an in vitro whole blood assay.

MAbs 2020 Jan-Dec;12(1):1715705

Biochemical and Cellular Pharmacology Department, Genentech Inc, South San Francisco, CA, USA.

For antibody-drug conjugates to be efficacious and safe, they must be stable in circulation to carry the payload to the site of the targeted cell. Several components of a drug-conjugated antibody are known to influence stability: 1) the site of drug attachment on the antibody, 2) the linker used to attach the payload to the antibody, and 3) the payload itself. In order to support the design and optimization of a high volume of drug conjugates and avoid unstable conjugates prior to testing in animal models, we wanted to proactively identify these potential liabilities. Therefore, we sought to establish an in vitro screening method that best correlated with in vivo stability. While traditionally plasma has been used to assess in vitro stability, our evaluation using a variety of THIOMAB antibody-drug conjugates revealed several disconnects between the stability assessed in vitro and the in vivo outcomes when using plasma. When drug conjugates were incubated in vitro for 24 h in mouse whole blood rather than plasma and then analyzed by affinity capture LC-MS, we found an improved correlation to in vivo stability with whole blood (R = 0.87, coefficient of determination) compared to unfrozen or frozen mouse plasma (R = 0.34, 0.01, respectively). We further showed that this whole blood assay was also able to predict in vivo stability of other preclinical species such as rat and cynomolgus monkey, as well as in human. The screening method utilized short (24 h) incubation times, as well as a custom analysis software, allowing increased throughput and in-depth biotransformation characterization. While some instabilities that were more challenging to identify remain, the method greatly enhanced the process of screening, optimizing, and lead candidate selection, resulting in the substantial reduction of animal studies.
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http://dx.doi.org/10.1080/19420862.2020.1715705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999835PMC
February 2021

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Bioorg Med Chem Lett 2020 02 18;30(4):126907. Epub 2019 Dec 18.

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.
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http://dx.doi.org/10.1016/j.bmcl.2019.126907DOI Listing
February 2020

2019 White Paper On Recent Issues in Bioanalysis: FDA BMV Guidance, ICH M10 BMV Guideline and Regulatory Inputs ( - Recommendations on 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and Regulatory Agencies' Input on Bioanalysis, Biomarkers and Immunogenicity).

Bioanalysis 2019 Dec 9;11(23):2099-2132. Epub 2019 Dec 9.

PPD, Richmond, VA, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA on 1-5 April 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on the 2018 FDA BMV guidance, 2019 ICH M10 BMV draft guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy. Part 1 (Innovation in small molecules and oligonucleotides and mass spectrometry method development strategies for large molecules bioanalysis) and Part 3 (New insights in biomarker assay validation, current and effective strategies for critical reagent management, flow cytometry validation in drug discovery and development and CLSI H62, interpretation of the 2019 FDA immunogenicity guidance and gene therapy bioanalytical challenges) are published in volume 10 of , issues 22 and 24 (2019), respectively.
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http://dx.doi.org/10.4155/bio-2019-0270DOI Listing
December 2019

Immunoaffinity LC-MS/MS to quantify a PEGylated anti-Factor D Fab biotherapeutic in cynomolgus monkey serum.

Bioanalysis 2019 Dec 8;11(23):2161-2173. Epub 2019 Nov 8.

Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

To develop a sensitive hybrid immunoaffinity LC-MS/MS monkey serum assay to quantify multiple components of anti-Factor D; a complex PEGylated Fab biotherapeutic explored as a therapy for age-related macular degeneration. Immunoaffinity enrichment of PEGylated anti-Factor D Fab, including fully conjugated, partially conjugated and unconjugated (i.e., free) Fab species, using a capture reagent coupled to magnetic beads was performed. The surrogate peptides derived from the therapeutic Fab via trypsin digestion were measured to obtain the total Fab concentrations. The method demonstrated the ability to accurately quantify both PEGylated and unconjugated Fab species. It was successfully validated with a LLOQ at 25.0 ng/ml.
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http://dx.doi.org/10.4155/bio-2019-0082DOI Listing
December 2019

High-Resolution Characterization of ADCs by Orbitrap LCMS.

Methods Mol Biol 2020 ;2078:213-219

Genentech Inc., South San Francisco, CA, USA.

Antibody-drug conjugates (ADCs) have complex molecular structures as they are composed of both small and large molecules, and they often undergo biotransformation over time in circulation. Here we describe a high-resolution Orbitrap MS approach for the characterization of ADC biotransformation and stability. Compared with conventional approach by Q-TOF MS, the method described here significantly improved the mass resolution and enabled more comprehensive characterization of ADC catabolites. It is particularly beneficial for characterizing ADC biotransformations with small mass changes.
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http://dx.doi.org/10.1007/978-1-4939-9929-3_14DOI Listing
January 2021

Gender effect of hyperuricemia on the development of nonalcoholic fatty liver disease (NAFLD): A clinical analysis and mechanistic study.

Biomed Pharmacother 2019 Sep 25;117:109158. Epub 2019 Jun 25.

Hangzhou Xixi Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, 310023, Zhejiang, China. Electronic address:

Background And Aims: Hyperuricemia is a risk factor for nonalcoholic fatty liver disease (NAFLD), however, the effect of gender on the hyperuricemia-related NAFLD development remains unclear. Here, we evaluated the clinical characteristics of NAFLD patients with hyperuricemia, and experimentally recapitulated this condition in male rats in order to gain insights on the possible impact of gender on the development of NAFLD in patients with hyperuricemia.

Methods: The clinical characteristics of 238 NAFLD patients, together with the impacts of hyperuricemia on the major parameters related to the development of NALFD were analysed. In animal studies, NAFLD with hyperuricemia was induced in male SD rats using high-yeast high-fat diet containing potassium oxonate. The impact of uric acids on liver pathology, and the expression patterns of key molecules involved in the development of NAFLD, including silent information regulator 1 (SIRT1), nuclear factor kappa B subunit p65 (NF-κB p65), fork-head box class O-3a (FOXO3a), androgen receptor (AR), and xanthine oxidase (XO) were analysed.

Results: Male NAFLD patients with hyperuricemia displayed more frequent and extensive liver injury than those in female patients. In male rats, hyperuricemia was associated with increased levels of insulin, alanine aminotransferase (ALT) and triglyceride (TG). At the molecular level, hyperuricemia was associated with decreased expression of SIRT1 and its phosphorylation, phosphorylation of FOXO3a, increased expression of AR and XO, and deacetylation of NF-κB P65.

Conclusions: Hyperuricemia is a compounding factor for NAFLD, particularly in males. The severer hepatic injury observed in male NAFLD patients may be attributed to the suppression of SIRT1 signalling induced by hyperuricemia.
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http://dx.doi.org/10.1016/j.biopha.2019.109158DOI Listing
September 2019

Role of identified RNA N6-methyladenosine methylation in liver.

Anal Biochem 2019 08 7;578:45-50. Epub 2019 May 7.

The Fourth Clinical Medicine School of Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China. Electronic address:

N6-Methyladenosine (mA) is the most abundant and important internal modification site of RNA methylation in viruses and eukaryotic. mA RNA methylation plays key roles in the regulation of post-transcriptional gene expression, including messenger RNA (mRNA), microRNA (miRNA) and long noncoding RNA (lncRNA). And mA methylation regulates the various aspects of RNA metabolism, including structure, maturation, stability, splicing, export, translation and decay. Liver is a vital metabolic and digestive organ in the pathophysiological processes. Recent studies suggested that mA RNA modification highly regulates hepatic function and development of liver diseases. Here, we aim to summarize the biological and clinical significance of mA modification in hepatic growth and hepatic disease including viral hepatitis, non-alcoholic fatty liver disease (NAFLD), and liver cancer.
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http://dx.doi.org/10.1016/j.ab.2019.05.005DOI Listing
August 2019

Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

Bioconjug Chem 2019 05 22;30(5):1356-1370. Epub 2019 Apr 22.

Genentech Inc. , 1 DNA Way , South San Francisco , California 94080 , United States.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00133DOI Listing
May 2019

Roles of Identified Long Noncoding RNA in Diabetic Nephropathy.

J Diabetes Res 2019 12;2019:5383010. Epub 2019 Feb 12.

The First Clinical Medical College, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075 Sichuan, China.

Diabetes mellitus is the leading chronic disease in the world, and diabetic nephropathy (DN) as one of its complications could increase the mortality. The development of DN is associated to abnormal hemodynamic factors like cytokine networks and the intervention of metabolic risk factors like blood pressure, blood glucose, and blood lipid. However, the pathogenesis of DN is still poorly understood. Although glucose-lowering drugs and insulins have significant effects on blood glucose, the fluctuation of blood glucose or other risk factors could continuously damage the kidney. Recent studies reported that the progression of DN is closely related to the expression of long noncoding RNA (lncRNA), which is important for the early diagnosis and targeted intervention of DN. In this review, we briefly summarize the published studies on the functions and potential mechanism of reported lncRNA in the regulation of DN.
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http://dx.doi.org/10.1155/2019/5383010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390257PMC
July 2019

Treating hyperuricemia related non-alcoholic fatty liver disease in rats with resveratrol.

Biomed Pharmacother 2019 Feb 14;110:844-849. Epub 2018 Dec 14.

The Hangzhou Xixi Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou 310023, Zhejiang, China. Electronic address:

Background Hyperuricemia is a recognised risk factor for the development of nonalcoholic fatty liver disease (NAFLD). This study aims to investigate the therapeutic effect of resveratrol (RES) on the treatment of hyperuricemia-related NAFLD in rats and the underlying mechanisms. Methods NAFLD with hyperuricemia was induced in rats using high-yeast high-fat diet containing potassium oxonate. The impact of RES on liver pathology, and the expression of silent information regulator 1 (SIRT1), fork-head box class O-3a (FOXO3a), and nuclear factor kappa B subunit p65 (NF-κB p65) was analysed. Results RES significantly improved liver histology and reversed serum biochemical abnormalities. At the molecular level, RES improved insulin resistance (IR), inhibited hepatic steatosis, reduced oxidative stress and liver inflammation, and these effects were likely mediated through SIRT1-mediated FOXO3a phosphorylation and NF-κB P65 deacetylation. Conclusions Resveratrol is a promising agent for the treatment of hyperuricemia-related NAFLD through activating SIRT1 pathways.
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http://dx.doi.org/10.1016/j.biopha.2018.12.039DOI Listing
February 2019

Optimizing hybrid LC-MS/MS binding conditions is critical: impact of biotransformation on quantification of trastuzumab.

Bioanalysis 2018 Oct 16. Epub 2018 Oct 16.

Department of BioAnalytical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.

Background: Hybrid ligand-binding (LB) LC-MS/MS protein quantitative assays involve a LB step for analyte enrichment that has less stringent requirements than the conventional LB assays.

Results: Herceptin™(trastuzumab) binding to HER2 extracellular domain was evaluated using on-bead and off-bead capture formats. The two formats yielded significantly different trastuzumab concentrations in human and monkey serum pharmacokinetic samples. Biotransformations, including deamidation of asparagine and isomerization of aspartic acid near the complementarity-determining regions of trastuzumab, had a profound impact on the LB step for analyte enrichment and trastuzumab quantification.

Conclusion: Quantitative measurements were profoundly impacted by LB conditions in a hybrid LB LC-MS/MS protein assay due to biotransformations. Therefore, similar to conventional LB assays, binding conditions should be carefully evaluated during assay development.
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http://dx.doi.org/10.4155/bio-2018-0196DOI Listing
October 2018

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia.

MAbs 2018 Nov-Dec;10(8):1312-1321. Epub 2018 Oct 2.

a Preclinical Translational Pharmacokinetics Department , Genentech Inc. , South San Francisco , CA , USA.

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.
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http://dx.doi.org/10.1080/19420862.2018.1517565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284592PMC
June 2019

Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody-Drug Conjugates.

Mol Pharm 2018 09 6;15(9):3979-3996. Epub 2018 Aug 6.

Wuxi Apptec , 288 Fute Zhong Road , Waigaoqiao Free Trade Zone, Shanghai 200131 , China.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00431DOI Listing
September 2018

New opportunities with quantification of protein therapeutics by LC-MS.

Bioanalysis 2018 Jul;10(13):971-973

Genentech Inc., 1 DNA Way, San Francisco, CA 94080, USA.

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http://dx.doi.org/10.4155/bio-2018-0151DOI Listing
July 2018

Characterization of in vivo biotransformations for trastuzumab emtansine by high-resolution accurate-mass mass spectrometry.

MAbs 2018 10 26;10(7):960-967. Epub 2018 Jul 26.

a Genentech Research and Early Development, Genentech Inc ., South San Francisco, CA , USA.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) designed for the treatment of HER2-positive cancers. T-DM1 is composed of the humanized monoclonal antibody trastuzumab connected to a maytansine derivative cytotoxic drug, via a nonreducible thioether linker at random lysine residues, and therefore has a very complex molecular structure. It was anticipated that T-DM1 undergoes biotransformations in circulation. However, there was limited knowledge on these structural changes due to bioanalytical challenges. Here, we have investigated the in vivo biotransformations of T-DM1 using a high-resolution accurate-mass (HR/AM) mass spectrometry approach. Three types of biotransformations were characterized for T-DM1 in circulation in tumor-bearing mice, including cysteine or glutathione adduct formation via maleimide exchange, loss of maytansinol via ester hydrolysis, as well as addition of HO via linker-drug hydrolysis. These results provide new insights into in vivo catabolism of T-DM1.
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http://dx.doi.org/10.1080/19420862.2018.1494487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204834PMC
October 2018

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC.

MAbs 2018 07 18;10(5):738-750. Epub 2018 May 18.

a Preclinical Translational Pharmacokinetics Department.

For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.
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http://dx.doi.org/10.1080/19420862.2018.1465160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150628PMC
July 2018

Conjugation of Indoles to Antibodies through a Novel Self-Immolating Linker.

Chemistry 2018 Apr 25;24(19):4830-4834. Epub 2018 Mar 25.

WuXi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, P. R. China.

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.
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http://dx.doi.org/10.1002/chem.201800859DOI Listing
April 2018

Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification.

Bioconjug Chem 2018 04 13;29(4):1155-1167. Epub 2018 Mar 13.

Genentech, Inc. , 1 DNA Way , South San Francisco , California 94080 , United States.

Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00785DOI Listing
April 2018

Intratumoral Payload Concentration Correlates with the Activity of Antibody-Drug Conjugates.

Mol Cancer Ther 2018 03 18;17(3):677-685. Epub 2018 Jan 18.

Drug Metabolism & Pharmacokinetics, Genentech Inc., South San Francisco, California.

Antibody-drug conjugates (ADC) have become important scaffolds for targeted cancer therapies. However, ADC exposure-response correlation is not well characterized. We demonstrated that intratumor payload exposures correlated well with the corresponding efficacies of several disulfide-linked ADCs, bearing an DNA alkylating agent, pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD), in HER2-expressing xenograft models. The correlation suggests that a threshold concentration of intratumor payload is required to support sustained efficacy and an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy ("Plateau" effect). In contrast to tumor PBD concentrations, related assessments of systemic exposures, plasma stability, and drug-to-antibody ratio changes of related ADCs did not consistently rationalize the observed ADC efficacies. A minimal efficacious dose could be determined by ADC dose-fractionation studies in the xenograft models. Mechanistic investigations revealed that both linker immolation and linker disulfide stability are the key factors that determine intratumor PBD concentrations. Overall, this study demonstrates how a linker design can impact ADC efficacy and that the intratumor exposure of a payload drug as the molecular mechanism quantitatively correlate with and predict the antitumor efficacy of ADCs. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0697DOI Listing
March 2018

Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity.

J Med Chem 2018 02 21;61(3):989-1000. Epub 2017 Dec 21.

Genentech, Inc. , 1 DNA Way, South San Francisco, California 94080, United States.

Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01430DOI Listing
February 2018