Principles of vaccine potency assays.

Compared with biologics, vaccine potency assays represent a special challenge due to their unique compositions, multivalency, long life cycles and global distribution. Historically, vaccines were released using in vivo potency assays requiring immunization of dozens of animals. Modern vaccines use a variety of newer analytical tools including biochemical, cell-based and immunochemical methods to measure potency. The choice of analytics largely depends on the mechanism of action and ability to ensure lot-to-lot consistency. Live vaccines often require cell-based assays to ensure infectivity, whereas recombinant vaccine potency can be reliably monitored with immunoassays. Several case studies are presented to demonstrate the relationship between mechanism of action and potency assay. A high-level decision tree is presented to assist with assay selection.

Development and Characterization of an HPV Type-16 Specific Modified DNA Aptamer for the Improvement of Potency Assays.

Measuring vaccine potency is critical for vaccine release and is often accomplished using antibody-based ELISAs. Antibodies can be associated with significant drawbacks that are often overlooked including lot-to-lot variability, problems with cell-line maintenance, limited stability, high cost, and long discovery lead times. Here, we address many of these issues through the development of an aptamer, known as a slow off-rate modified DNA aptamer (SOMAmer), which targets a vaccine antigen in the human papillomavirus (HPV) vaccine Gardasil. The aptamer, termed HPV-07, was selected to bind the Type 16 virus-like-particle (VLP) formed by the self-assembling capsid protein L1. It is capable of binding with high sensitivity (EC50 of 0.1 to 0.4 µg/mL depending on assay format) while strongly discriminating against other VLP types. The aptamer competes for binding with the neutralizing antibody H16.V5, indicating at least partial recognition of a neutralizing and clinically relevant epitope. This makes it a useful reagent for measuring both potency and stability. When used in an ELISA format, the aptamer displays both high precision (intermediate precision of 6.3%) and a large linear range spanning from 25% to 200% of a typical formulation. To further exploit the advantages of aptamers, a simplified mix and read assay was also developed. This assay format offers significant time and resource reductions compared to a traditional ELISA. These results show aptamers are suitable reagents for biological potency assays, and we expect that their implementation could improve upon current assay formats.

Evaluation of two ELISA methods to detect therapeutic anti-IGF1R antibodies in clinical study samples of dalotuzumab.

BACKGROUND: Dalotuzumab (MK 0646), an anti-IGF1R antibody intended for cancer therapy, has progressed to Phase III clinical trials. To evaluate pharmacokinetic properties, we developed and compared two ELISAs to measure dalotuzumab in human serum and validated the second method following regulatory guidelines for ligand-binding assays. RESULTS: After an IGF1R-mediated capture step, dalotuzumab was detected by either an antihuman IgGFc- or by an antihuman IgG1-specific antibody. The assay range was 20 to 2000 ng/ml with mean inter-day accuracy of controls ranging from 97 to 108% (method A) and 83 to 97% (method B), respectively. Mean assay precision was ≤20% CV both intra- and inter-day. Other parameters that were validated included dilution linearity, stability, interferences and incurred sample reanalysis. In addition, application of both assay formats to clinical sample analysis was demonstrated establishing time-concentration curves. CONCLUSION: As the methods rely on commercial reagents, they may be applicable to other anti-IGF1R antibodies and facilitate the development of new therapeutics.

Comparison of the NIDS® rapid assay with ELISA methods in immunogenicity testing of two biotherapeutics.

INTRODUCTION: Rapid lateral flow immunogenicity assays for the detection of anti-drug antibodies (ADAs) to two biotherapeutic antibodies, an anti-HER2 antibody and an anti-TNF-α antibody, were developed using ANP Technologies, Inc.'s proprietary Nano-Intelligent Detection System (NIDS®) and compared to their ELISA counterparts. METHODS: Biotin and hapten-labeled drugs are incubated with the patient serum sample to allow ADA to form a bridge complex with each drug conjugate. The reaction mixture is then added to a test strip with an anti-hapten capture zone which captures the mixed bridge complex. The bridge-complexed biotinylated drug then reacts with streptavidin-labeled gold particles in situ. The signal developed at the capture zone, which is directly proportional to ADA in the sample, is then quantitatively measured with a handheld reader. The counterpart ELISAs were run using the same reagents. Dose-response, specificity/free drug depletion, and screening cut-point assays were performed using both methods. RESULTS: The rapid assays' performance compare very closely to their ELISA counterparts'. Both types of assays identified the same positive samples in screening a limited population of 50 normal serum samples for the anti-HER2 antibody. In the case of anti-TNF-α, both assays identified the same positive samples out of 50 normal and 20 rheumatoid arthritis patient serum samples but differed in the assessment of two others. The rapid assay correctly identified as negative an ELISA false positive sample, and correctly tested as positive an ELISA false negative sample. Positive results were verified with a specificity/free drug depletion assay. DISCUSSION: The NIDS® rapid immunogenicity assay offers distinct advantages over current methods in simplicity, low cost, and short time to result. More importantly, the method requires no sample dilution and no washing steps which can perturb fragile complexes formed by low-affinity ADAs. Thus, the assay can potentially detect ADAs with various affinities.

Application of miniaturized immunoassays to discovery pharmacokinetic bioanalysis.

INTRODUCTION: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays. METHODS: Three representative example assays--a generic anti-human IgG, a target specific and an anti-drug capture assay--were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support. RESULTS: Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10µL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less. DISCUSSION: Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies.

Pharmacokinetic immunoassay methods in the presence of soluble target.

Soluble targets represent a special challenge when employing ligand binding assays to support pharmacokinetic analysis of monoclonal therapeutics. Target-engaged antibody is not available for binding in immunoassays employing anti-idiotype-specific antibodies or target for capture. We investigated several formats of total antibody assays that show reduced interference of soluble targets: direct target capture, indirect target capture and acid dissociation. While indirect target capture worked well for a regular affinity antibody against DKK1, a high affinity antibody against PCSK9 required an additional acid dissociation step. The choice of a suitable format was antibody and target dependent. Our results offer several choices to approach immunoassay development for soluble targets.

Protein profiling of pleural effusions to identify malignant pleural mesothelioma using SELDI-TOF MS.

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.

Anti-mesothelin antibodies and circulating mesothelin relate to the clinical state in ovarian cancer patients.

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 > or = 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 > or = 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.

The use of multiple novel tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass.

OBJECTIVES: The CA125 tumor marker is used to help predict the presence of ovarian cancer in patients with an adnexal mass. Because elevated CA125 levels occur in many benign gynecologic conditions, we set out to identify other novel biomarkers that would increase the sensitivity and specificity of CA125. METHODS: Serum and urine samples were obtained preoperatively from women undergoing surgery for an adnexal mass. The samples were analyzed for levels of CA125, SMRP, HE4, CA72-4, activin, inhibin, osteopontin, epidermal growth factor (EGFR), and ERBB2 (Her2) and were compared to final pathology results. Logistic regression models were estimated for all markers and combinations, with cross-validation analysis performed to obtain the sensitivities at set specificities of 90%, 95%, and 98%. RESULTS: Two hundred and fifty-nine patients with adnexal masses were enrolled. Of these, 233 patients were eligible for analysis with 67 invasive epithelial ovarian cancers and 166 benign ovarian neoplasms. Mean values for all marker levels except Her2 differed significantly between patients with benign masses and cancer. As a single marker, HE4 had the highest sensitivity at 72.9% (specificity 95%). Comparatively, combined CA125 and HE4 yielded the highest sensitivity at 76.4% (specificity 95%), with additional markers adding minimally to the sensitivity of this combination. HE4 was the best single marker for Stage I disease, with no increase in sensitivity when combined with CA125 or any other marker. CONCLUSIONS: As a single tumor marker, HE4 had the highest sensitivity for detecting ovarian cancer, especially Stage I disease. Combined CA125 and HE4 is a more accurate predictor of malignancy than either alone.

MESOMARK: a potential test for malignant pleural mesothelioma.

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, 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 degrees C and upon storage for at least 7 days at 2-8 degrees 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 asbestos-exposed individuals, respectively. Concentrations in other nonmalignant and malignant conditions were similar to those in healthy controls. CONCLUSIONS: The MESOMARK assay is analytically robust and may be useful for the detection and management of mesothelioma.

MESOMARK(®) in vitro diagnostic test for mesothelioma.

Mesothelioma is a highly progressive tumor with a poor prognosis. In this article, the authors give a thorough introduction into and evaluation of the MESOMARK(®) in vitro test - the only blood test for the management of patients with mesothelioma approved by the US FDA. In Europe, Australia, New Zealand and Canada, the test is approved or licensed for the measurement of the soluble mesothelin, also termed as soluble mesothelin-related peptides. In the US, it is approved for the monitoring of patients with epithelioid and biphasic malignant mesothelioma.

Mesothelin variant 1 is released from tumor cells as a diagnostic marker.

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 approximately 40 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.

Listeria monocytogenes-based antibiotic resistance gene-free antigen delivery system applicable to other bacterial vectors and DNA vaccines.

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.

Immunization with a plant-produced colorectal cancer antigen.

Cancer vaccination has become an important focus of oncology in recent years. Active immunization with tumor-associated antigens such as colorectal cancer antigen GA733-2 is thought to potentially overcome the reoccurrence of metastasis. As recombinant protein production in bioreactors is costly and subject to growing safety concerns, we tested plants as an alternative for the expression of a potential colorectal cancer vaccine. Comparing colorectal cancer antigen GA733-2 produced in tobacco plants with the same antigen produced in insect cell culture, we found a similar humoral immune response to injection of either of the two antigen preparations into mice. Some minor differences were observed in the cellular response that might be due to impurities. Our studies compare for the first time, immunization with the same antigen expressed in either plants or insect cell culture. This will provide important data for use of plants as production systems of therapeutics.