Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for Therapeutic Studies.

Ulcerative colitis is a chronic inflammatory disease affecting the colon and is characterized by epithelial damage and barrier dysfunction. Upregulation of the tight junction protein claudin-2 by cytokines is hypothesized to contribute to the dysregulation of the epithelial barrier. New therapeutic agents which block the action of cytokines are being investigated in patients with ulcerative colitis. In order to understand the potential of these therapies, it is important to have reliable assays that can assess downstream endpoints that reflect drug mechanism of action. The aim of the current study was therefore to establish & validate an assay to reproducibly assess the expression and distribution of claudin-2 in human colon biopsy samples. Initially, the potential to measure claudin-2 protein by immunohistochemistry (IHC) was investigated. To identify suitable reagents to develop an IHC assay, pre-established criteria were used to screen five commercial antibodies by Western blotting, immunofluorescence and immunohistochemistry on claudin-2 positive and negative cells and healthy and ulcerative colitis colon tissue. Despite some of these antibodies specifically detecting claudin-2 using some of these techniques, none of the antibodies showed the expected specific staining pattern in formalin fixed human colon samples. As an alternative method to detect claudin-2 expression and distribution in formalin fixed biopsy sections, an in situ hybridization assay was developed. This assay underwent a novel tiered approach of validation to establish that it was fit-for-purpose, and suitable for clinical deployment. In addition, to understand the possible relationship of claudin-2 in the context of disease severity, expression was compared to the Geboes score. Overall, the microscopical Geboes score correlated with the claudin-2 biomarker score for samples that retained crypt morphology; samples with the highest Geboes score were not specifically distinguished, probably due to crypt destruction. In summary, we have applied a strategy for identifying target-specific antibodies in formalin fixed biopsy samples and highlighted that (published) antibodies may not correctly identify the intended antigen in tissues fixed using this method. Furthermore, we have developed and, for the first time, validated an in situ hybridization assay for detection of claudin-2 mRNA, suitable for use as a supportative method in clinical trials. Using our validated assay, we have demonstrated that increased claudin-2 expression correlates with the severity of ulcerative colitis, where crypt destruction is not seen.

The Use of Antibodies in Small-Molecule Drug Discovery.

Antibodies are powerful research tools that can be used in many areas of biology to probe, measure, and perturb various biological structures. Successful drug discovery is dependent on the correct identification of a target implicated in disease, coupled with the successful selection, optimization, and development of a candidate drug. Because of their specific binding characteristics, with regard to specificity, affinity, and avidity, coupled with their amenability to protein engineering, antibodies have become a key tool in drug discovery, enabling the quantification, localization, and modulation of proteins of interest. This review summarizes the application of antibodies and other protein affinity reagents as specific research tools within the drug discovery process.

Identification of a subset of human non-small cell lung cancer patients with high PI3Kß and low PTEN expression, more prevalent in squamous cell carcinoma.

PURPOSE: The phosphoinositide 3-kinase (PI3K) pathway is a major oncogenic signaling pathway and an attractive target for therapeutic intervention. Signaling through the PI3K pathway is moderated by the tumor suppressor PTEN, which is deficient or mutated in many human cancers. Molecular characterization of the PI3K signaling network has not been well defined in lung cancer; in particular, the role of PI3Kß and its relation to PTEN in non-small cell lung cancer NSCLC remain unclear. EXPERIMENTAL DESIGN: Antibodies directed against PI3Kß and PTEN were validated and used to examine, by immunohistochemistry, expression in 240 NSCLC resection tissues [tissue microarray (TMA) set 1]. Preliminary observations were extended to an independent set of tissues (TMA set 2) comprising 820 NSCLC patient samples analyzed in a separate laboratory applying the same validated antibodies and staining protocols. The staining intensities for PI3Kß and PTEN were explored and colocalization of these markers in individual tumor cores were correlated. RESULTS: PI3Kß expression was elevated significantly in squamous cell carcinomas (SCC) compared with adenocarcinomas. In contrast, PTEN loss was greater in SCC than in adenocarcinoma. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC in TMA set 1 with higher PI3Kß and lower PTEN expression when compared with adenocarcinoma. These findings were reinforced following independent analyses of TMA set 2. CONCLUSIONS: We identify for the first time a subset of NSCLC more prevalent in SCC, with elevated expression of PI3Kß accompanied by a reduction/loss of PTEN, for whom selective PI3Kß inhibitors may be predicted to achieve greater clinical benefit.

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα.

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.

Validation of the BRCA1 antibody MS110 and the utility of BRCA1 as a patient selection biomarker in immunohistochemical analysis of breast and ovarian tumours.

BRCA1 protein measurement has previously been evaluated as a potential diagnostic marker without reaching a conclusive recommendation. In this study, we applied current best practice in antibody validation to further characterize MS110, a widely used antibody targeting BRCA1. Antibody specificity was investigated using different biochemical validation techniques. We found that BRCA1 could not be reliably detected using immunoprecipitation and Western blot in endogenously expressing cells. We used immunohistochemistry on formalin-fixed paraffin-embedded cell pellets to establish compatibility with formalin-fixed paraffin-embedded samples. We demonstrated that in transfected cells and cell lines with known genetic BRCA1 status, MS110 successfully detected BRCA1 giving the expected level of staining in immunohistochemistry. Following this, we investigated the use of BRCA1 protein measurement by immunohistochemistry in a cohort of triple negative breast and serous ovarian tumour samples to explore the use of BRCA1 protein measurement by immunohistochemistry for patient stratification. Using MS110 in repeated standardized experiments, on serial sections from a panel of patient samples, results demonstrated considerable run-to-run variability. We concluded that in formalin-fixed tissue samples, MS110 does detect BRCA1; however, using standard methodologies, BRCA1 expression levels in tissue samples is incompatible with the use of this protein as a statistically robust patient selection marker in immunohistochemistry. These results demonstrate the need for further development to deliver BRCA1 protein quantification by immunohistochemistry as a patient stratification marker.

Elevated MARK2-dependent phosphorylation of Tau in Alzheimer's disease.

The appearance of neurofibrillary tangles (NFT), one of the major hallmarks of Alzheimer's disease (AD), is most likely caused by inappropriate phosphorylation and/or dephosphorylation of tau, eventually leading to the accumulation of NFTs. Enhanced phosphorylation of tau on Ser(262) is detected early in the course of the disease and may have a role in the formation of tangles. Several kinases such as microtubule-affinity regulating kinase (MARK), protein kinase A, calcium calmodulin kinase II, and checkpoint kinase 2 are known to phosphorylate tau on Ser(262) in vitro. In this study, we took advantage of the in situ proximity ligation assay to investigate the role of MARK2, one of the four MARK isoforms, in AD. We demonstrate that MARK2 interacts with tau and phosphorylates tau at Ser(262) in stably transfected NIH/3T3 cells expressing human recombinant tau. Staurosporine, a protein kinase inhibitor, significantly reduced the interaction between MARK2 and tau, and also phosphorylation of tau at Ser(262). Furthermore, we observed elevated interactions between MARK2 and tau in post-mortem human AD brains, compared to samples from non-demented elderly controls. Our results from transfected cells demonstrate a specific interaction between MARK2 and tau, as well as MARK2-dependent phosphorylation of tau at Ser(262). Furthermore, the elevated interactions between MARK2 and tau in AD brain sections suggests that MARK2 may play an important role in early phosphorylation of tau in AD, possibly qualifying as a therapeutic target for intervention to prevent disease progression.