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Background: Soluble mesothelin-related peptides (SMRP) have been reported to be potential biomarkers for malignant pleural mesothelioma (MPM). We report analytical and preliminary clinical studies of MESOMARK TM , a quantitative assay for SMRP. Methods: The MESOMARK assay is a 2-step immunoenzymatic assay in an ELISA format with a 6-point calibration curve (0 -32 nmol/L). We assessed analytical imprecision, analyte stability, and analytical interferences. We measured SMRP by this assay in 409 apparently healthy individuals (reference interval study), 177 patients with nonmalignant conditions, and 500 cancer patients, including 88 with MPM. Results: The limit of detection was 0.16 nmol/L. At 2-19 nmol/L, intraassay imprecision (CV) was 1.1%-5.3%, and total imprecision was 4.0%-11.0%. The mean dilution recovery for 5 samples was 109% (range, 99%-113%). No interference was seen from added bilirubin (200 mg/L), hemoglobin (500 mg/L), triglycerides (30 g/L), chemotherapeutic agents, or other tested substances. Recombinant mesothelin was stable in serum upon freeze/thaw at ؊70°C and upon storage for at least 7 days at 2-8°C. The 99 th percentile of the reference group was 1.5 nmol/L [95% confidence interval (CI), 1.2-1.6 nmol/L; n ‫؍‬ 409], and mean SMRP was significantly higher in sera from patients with MPM (7.5 nmol/L; 95% CI, 2.8 -12.1 nmol/L; n ‫؍‬ 88). SMRP was increased in 52% and 5% of MPM patients and asbestosexposed individuals, respectively. Concentrations in other nonmalignant and malignant conditions were similar to those in healthy controls.

The mesothelin family comprises (at least) three variants and includes the precursor for megakaryocyte potentiating factor (MPF). Assaying soluble mesothelin-related protein (SMRP) molecules in serum and other body fluids from patients with certain cancers can provide diagnostically useful information. We have constructed fusion proteins of mesothelin variants 1, 2, and 3, made monoclonal antibodies, and investigated the binding specificity of these and three previously generated monoclonal antibodies to each of the three mesothelin variants. According to flow cytometry, the molecule that is most frequently expressed at the surface of cells from ovarian carcinomas and certain other tumors is mesothelin variant 1. Similarly, SMRP released into ascites from a patient with ovarian carcinoma was shown to have a molecular weight of f40 kDa and, according to sequencing, to be variant 1. A published sandwich ELISA was shown to detect variants 1 and 3 and to be much more sensitive than a newly constructed ELISA, which detects only variant 3, the former being positive in 28 of 41 (68%) sera from patients with ovarian cancer as compared with 6 of 41 sera (15%). A standard curve was constructed to measure SMRP with a limit of detection of 200 pg/mL to facilitate future quantitative studies. (Cancer Epidemiol Biomarkers Prev 2006;15(5):1014 -20)

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Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels. In vaccine development, with live vaccine vectors, this allows greater flexibility and the ability to induce larger antigen amounts through multiple gene copies. However, plasmid retention often requires antibiotic resistance markers, the presence of which has been discouraged in clinical applications by the Food and Drug Administration. Therefore, we developed a Listeria monocytogenes-Escherichia coli shuttle plasmid that is retained by complementation of D-alanine racemase-deficient mutant strains both in vitro and in vivo. Our technology potentially allows the production of antibiotic resistance marker-free DNA vaccines as well as bacterial vaccine vectors devoid of engineered antibiotic resistances. As a proof of concept, we applied the D-alanine racemase complementation system to our Listeria cancer vaccine platform. With a transplantable tumor model, we compared the efficacy of the new Listeria vector to that of an established vector containing a conventional plasmid carrying a tumor-specific antigen. Both vaccine vector systems resulted in long-term regression of established tumors, with no significant difference between them. Thus, the Listeria vaccine vector presented here potentially complies with Food and Drug Administration regulations and could be developed further for clinical use.The revolution in genetic engineering over the last two decades has allowed the development of recombinant DNA vaccines and bacterial antigen delivery systems, opening up new horizons in immunization against infectious diseases and even cancer. Recombinant antigens can be presented to the immune system after immunization with DNA directly or with bacterial vectors expressing vaccine antigens. Both approaches have the advantages of potentially low per-dose costs, ease of distribution and storage, and rational vaccine design. However, the required molecular engineering techniques often rely on the use of antibiotic resistance markers for retention of the recombinant gene. For example, the production of plasmid-based DNA vaccines in Escherichia coli usually relies on kanamycin resistance, which ensures plasmid stability during culture in the presence of the antibiotic. Bacterial delivery systems carrying plasmids also contained antibiotic resistance markers for in vitro selection in addition to in vivo retention systems (7,(11)(12)(13)26). Although the use of plasmids could sometimes be circumvented by integrating the recombinant gene into the bacterial genome by homologous recombination, this process is tedious and time-consuming, resulting in reduced vaccine flexibility.A recent accelerated chromosomal integration technology developed for Listeria spp. relied again on the use of antibiotic markers (20). The Food and Drug Administration, however, discouraged the in vivo use of genes transferring antibiotic resistance in several communiqués because of the associated potential risk of environmental spread (9...

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3–7 April 2017 with participation of close to 750 professionals 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 and immunogenicity. 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 analysis involving LC–MS, hybrid ligand-binding assay (LBA)/LC–MS and LBA approaches. This 2017 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC–MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC–MS for biotherapeutics and regulatory agencies’ inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

Most human ovarian carcinomas express mesothelin, which is shed as a diagnostically useful biomarker. We applied an ELISA to measure antibodies to native mesothelin in serum from a series of patients with divergent clinical outcomes. The level of anti-mesothelin antibodies determined as OD 450 nm and referred to as absorption units (AU) for 1:20 diluted serum was higher in patients who remained disease-free after therapy [no evidence of disease (NED); n = 14] than in patients whose disease recurred [clinical evidence of disease (CED); n = 21; P < 0.01]. Applying AU z 0.5 at a serum dilution of 1:20 as cutoff, 10 of 14 (71%) ovarian carcinoma patients with NED and 9 of 21 (43%) patients with CED had antibodies to mesothelin compared with 6 of 23 (26%) healthy women (P < 0.008) and 5 of 24 (21%) women with other benign gynecologic diseases (P < 0.003), whereas 7 of 9 (78%) of women with pelvic inflammatory disease were positive. Three of the 14 (21%) NED patients had circulating mesothelin detected as an AU z 0.2 at a serum dilution of 1:40 (P < 0.005) compared with 15 of 21 (71%) CED patients, and 9 of 14 (64%) NED patients (P < 0.0002) were positive for antibodies and negative for antigen compared with 1 of 21 (5%) CED patients. Although our data indicate that an antibody response to mesothelin is an important correlate of ovarian carcinoma, prospective studies are needed to show whether the measurement of such antibodies (alone or together with antigen) aids the diagnosis and monitoring of patients. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1520 -6)

Diagnosis of malignant pleural mesothelioma (MM) is limited. Novel proteomic techno_logies can be utilized to discover changes in expression of pleural proteins that might have diagnostic value. The objective of this study was to detect protein profiles that could be used to identify malignant pleural mesothelioma with surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Pleural effusions were collected from patients with confirmed mesothelioma (n = 41) and from patients with effusions due to other causes ([n = 48] cancerous and non-cancerous). Samples were fractionated using anion exchange chromatography and bound to different types of ProteinChip array surfaces. All samples were also subjected to other commercially available immunoassays (human epididymes protein 4 [HE4], osteopontin [OPN], soluble mesothelin-related proteins [SMRP], and the cytokeratin 19 fragment [CYFRA 21-1]). Peak intensity data obtained by SELDI-TOF were subjected to classification algorithms in order to identify potential classifier peaks. A protein peak at m/z 6614 was characterized as apolipoprotein (Apo) CI. In this setting, the sensitivity and specificity of this potential biomarker was 76 % and 69 %, respectively. The area under the receiver operating characteristic curve (AUC) for Apo CI was 0.755, thereby outperforming OPN, HE4, and CYFRA 21-1. SMRP performed best with an AUC of 0.860 with a sensitivity of 83% and specificity of 74%. Our study validates the use of SMRP as a diagnostic marker for pleural mesothelioma and furthermore suggests that Apo CI levels could be used in the future to discriminate pleural mesothelioma from other causes of exudates.