Affimers as anti-idiotypic affinity reagents for pharmacokinetic analysis of biotherapeutics.

Therapeutic antibodies are the fastest growing class of drugs in the treatment of cancer, and autoimmune and inflammatory diseases that require the concomitant development of assays to monitor therapeutic antibody levels. Here, we demonstrate that the use of Affimer nonantibody binding proteins provides an advantage over current antibody-based detection systems. For four therapeutic antibodies, we used phage display to isolate highly specific anti-idiotypic Affimer reagents, which selectively bind to the therapeutic antibody idiotype. For each antibody target the calibration curves met US Food and Drug Administration criteria and the dynamic range compared favorably with commercially available reagents. Affimer proteins therefore represent promising anti-idiotypic reagents that are simple to select and manufacture, and that offer the sensitivity, specificity and consistency required for pharmacokinetic assays.

A place for precision medicine in bladder cancer: targeting the FGFRs.

Bladder tumors show diverse molecular features and clinical outcome. Muscle-invasive bladder cancer has poor prognosis and novel approaches to systemic therapy are urgently required. Non-muscle-invasive bladder cancer has good prognosis, but high recurrence rate and the requirement for life-long disease monitoring places a major burden on patients and healthcare providers. Studies of tumor tissues from both disease groups have identified frequent alterations of FGFRs, including mutations of FGFR3 and dysregulated expression of FGFR1 and FGFR3 that suggest that these may be valid therapeutic targets. We summarize current understanding of the molecular alterations affecting these receptors in bladder tumors, preclinical studies validating them as therapeutic targets, available FGFR-targeted agents and results from early clinical trials in bladder cancer patients.

High-content, high-throughput screening for the identification of cytotoxic compounds based on cell morphology and cell proliferation markers.

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.

A high-throughput assay to identify modifiers of premature chromosome condensation.

Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detected by DAPI staining using an Operetta imaging system. PCC and nuclei number were determined using Columbus analysis software. Two batches of nine plates were used to determine assay efficacy. Each plate contained four negative (nontargeting) and eight positive control siRNAs. Mean % PCC was 12.35% (n = 72) for negative controls and 4.25% (n = 144) for positive controls. Overall false-positive and false-negative rates were 0% (n = 72) and 2.1% (n = 144), respectively. This assay is currently being used to screen a human druggable genome siRNA library to identify novel therapeutic targets for cancer treatment. The assay can also be used to identify novel compounds and genes that induce PCC.