Fully international system of units-traceable protein quantification: what we can really measure
Abstract
Measurement is an indispensable methodology to find, prove, compare, understand, and develop scientific issues with regardless of field. The purpose of the measurement is to make an objective comparison through the quantification of objects, and measurement traceability and uncertainty are what is absolutely necessary for this. The traceability refers to a comparability of data, and the uncertainty indicates the degree of reliability. Protein is a key biological molecule, and is leading biotechnology and pharmaceutical, medical industry. Obviously, we are measuring the protein in a number of approaches. However, it is not clear to discuss about the accuracy of the results without any clear definition of measurand in those measurement. Accurate and reliable protein quantification is especially important in clinical analysis for diagnosis and therapeutic dose control for treatments. Based on the establishment of measurement traceability, the results can compare between them. As no analytical method can yet directely quantify whole proteins to the desired level of accuracy, proteins are reduced to analuzable entities, either peptide, amino acid, or element, which are then analyzed to deduce the quantity of original protein. An element and amino acid based reductive approaches provide an effective emans of relizing International System of Units (SI) traceability for pure-protein standards. These methods can apply to certification of peptide and protein standards for quantification of target proteins in mixture and clinical matrices samples. In this presentation, the development of primary measurement procedure for protein quantification, as well as the international collaborations for standardization of protein analysis will be discussed with recombinant human growth hormone, hemoglobin, and insulin as a model protein.
Development of a Novel Internalizing Human Antibody Targeting
Abstract
Epithelial ovarian cancer (EOC) invasion and metastasis are complex phenomena that result from the coordinated action of many metastatic regulators and must be overcome to improve clinical outcomes for patients with these cancers. The identification of novel therapeutic targets is critical because of the limited success of current treatment regimens, particularly in advanced-stage ovarian cancers. In this study, we found that tetraspanin 8 (TSPAN8) is overexpressed in about 52% (14/27) of EOC tissues and correlates with poor survival. Using small interfering RNA-mediated TSPAN8 knockdown and a competition assay with purified TSPAN8 large extracellular loop (TSPAN8-LEL) protein, we identified TSPAN8-LEL as a key regulator of EOC cell invasion. Furthermore, monotherapy with TSPAN8-blocking antibody we developed shows that antibody-based modulation of TSPAN8-LEL can significantly reduce the incidence of EOC metastasis without severe toxicity in vivo. Finally, we demonstrated that the TSPAN8-blocking antibody promotes the internalization and concomitant downregulation of cell surface TSPAN8. Collectively, our data suggest TSPAN8 as a potential novel therapeutic target in EOCs and antibody targeting of TSPAN8 as an effective strategy for inhibiting invasion and metastasis of TSPAN8-expressing EOCs.
Current status and challenges of 'antibody-drug conjugate(ADC)' for anti-cancer therapy
Abstract
Antibody drug conjugates (ADCs) are a new form of targeted therapy to overcome cancers, consisting of an antibody, a conditionally stable linker, and a cytotoxic drug. The concept of antibody-drug conjugate had been around for several decades, in which antibody serves as targeting agent and cytotoxic drug serves as actual killing player. However, it took several ‘success-failure’ tides until US FDA approved the first ADC product, Mylotarg, gemtuzumab ozogamicin, in 2000, which was withdrawn from the market a decade later owing to a lack of improvement in overall survival. A truly successful ADC drug, Adcetris (brentuximab vedotin) was generated from ‘Seattle Genetics’ and approved from US FDA for the treatment of Hodgkin’s and anaplastic large-cell lymphoma (ALCL). More recently, Kadcyla (Adotratuzumab emtansine, T-DM1) from Genentech was developed and FDA-approved for the breast cancer treatment. Despite the simple concept of components’ functions, the development of ADC has a lot of parameters to consider to make an effective ADC drug, which provide many hurdles to academia researchers and pharma developers. This talk will cover the key parameters, the limitations and current issues in the development of ADC drugs.
College of Medicine, National Cheng Kung University
Title
Antibodyome analysis using proteome microarrays
Abstract
Proteome microarrays contains thousands of proteins in one chip, which provides hundreds of thousands of antigenic epitopes for analyzing serological antibodies with a sample volume of less than 1 μl . By knowing the differences of serological antibodies between diseases and healthy controls, many diseases are able to be diagnosed with a simple blood test. Here, three examples using E. coli proteome microarrays are presented: bipolar disorder (BD), Kawasaki disease (KD) and Pre-eclampsia. In the case of BD, the plasmas of HCs and patients with bipolar I disorder in acute mania (BD-A) along with remission (BD-R) were collected. The initial samples consisting of 19 BD-A, 20 BD-R, and 20 HCs were probed with the microarrays. After selecting protein hits that recognized the antibody differences between BD and HC, the proteins were purified to construct BD focus arrays for training diagnosis committees and validation. Additional six BD-A, six BD-R, six HCs, and nine schizophrenic disorder (SZ, as another psychiatric control) samples were individually probed with the BD focus arrays and trained to form a diagnosis panel. In the single blind test using another four BD-A, four HC, and four SZ samples, the committee of BD-A versus HC was able to classify BD-A versus HC and SZ with 75% sensitivity and 80% specificity and 79% accuracy. In the case of Pre-eclampsia, the Escherichia coli chip was probed with samples from 29 individuals (16 pre-eclamptic women and 13 healthy pregnant women) to profile plasma antibodies. An antibody classifier was identified using k-top scoring pairs and additional samples for a blinded test were collected. The findings indicated that pre-eclamptic women developed more immunoglobulin G but less immunoglobulin M against bacterial surface proteins compared with healthy women. The k-top scoring pairs identified five pairs of immunogenic proteins as classifiers with a high accuracy of 90% in the blind test. In the case of Kawasaki disease (KD), plasmas were collected from KD patient before intravenous immunoglobulin treatment (KD1), at least 3 weeks after treatment (KD3), nonfever control (NC), and fever control (FC) children. The initial screening, which consisted of 20 KD1, 20 KD3, 20 NC, and 20 FC, were explored using E. coli proteome microarrays. About ∼70 proteins were shown to have high accuracy, e.g.0.78∼0.92, with regard to separating KD1, KD3, NC, and FC. Those proteins were then purified to fabricate KD focus arrays for training (n = 20 each) and blind-testing (n = 20 each). It only took 125 pl of plasma, less than a drop of blood, in the focus array assays. The AUC scores for blind tests of KD1 versus NC (17 protein markers), KD1 versus FC (20 protein markers), KD3 versus NC (9 protein markers), and KD1 versus KD3 (6 protein markers) were 0.84, 0.75, 0.99 and 0.98, respectively. These examples have demonstrated that the E. coli proteome microarray is a strong and robust tool for profiling antibodies and capable of screening disease plasma antibody differences.
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