One new technique that is providing unparalleled proteomic insight is a customized, high-density protein microarray, known as NAPPA (Nucleic Acid-Programmable Protein Array). NAPPA is poised to revolutionize high-throughput investigations of protein interactions and is being applied in the discovery of biomarkers—molecular indicators that can provide clinicians with early warning signs for a range of diseases. The NAPPA technology will augment other new proteomic techniques, including cell-based research. The following projects now underway at the Piper Center give a sense of the power and versatility of high-throughput technology for the study of issues critical to personalized diagnostic medicine.
New preclinical evidence indicates that cancer, including breast cancer, is closely monitored by the body’s immune system. This close surveillance—carried out by both the innate and the adaptive immune systems, detects subtle alterations to the proteome, including changes in protein expression, mutations, folding, glycosylation, and degradation. Localized immune responses to tumor antigens appear in draining lymph nodes before entering the systemic circulation.
Tumor antigens like the p53 protein are stable, robust and easily detected in serum, making such antibody responses highly attractive as biomarkers for disease. The problem for clinical application has been that antibodies display limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their practical application in cancer diagnosis. Further, autoimmune antibodies may occur in a limited subset of breast cancer patients.
If new autoantigens can be found, multivariant screening would be possible with significantly improved sensitivity and specificity of detection. Use of Nucleic Acid-Programmable Protein Array (NAPPA) allows researchers to forego the complex process of identifying purified proteins, making use instead of plasmid DNA.
Through this process, direct manipulation of the proteins is minimized and protein production can be carried out just-in-time for the experiment, avoiding issues of protein purification and stability. Furthermore, NAPPA allows researchers to look at thousands of proteins at one time, as they are produced simultaneously in situ, with remarkably consistent protein levels displayed.
The overall goal of this important work is to identify autoantibody biomarkers in blood that can be clinically applied for the early detection of cancers. Blood samples from health control subjects are being compared with those of cancer patients in order to find autoantibodies that are patient-specific. While our first experimental efforts focus on breast cancer, research is quickly expanding to include other diseases, including ovarian cancer, prostate cancer and Lung Cancer.
Type 1 diabetes is a serious autoimmune disorder, usually resulting from a combination of environmental and hereditary factors. It is related to a defect in immune surveillance that compromises self-tolerance to pancreatic islet antigens and results in the selective targeting, destruction and progressive loss of beta cells. Over time, this process acts to impair the body’s ability to secrete insulin and maintain blood glucose homeostasis. If left untreated, the disorder is often fatal.
Diabetes affects 10-20 million people worldwide, often seriously altering the lifestyle of those afflicted and pre-disposing them to many secondary disease complications, often severe. Humoral auto-antibodies to pancreatic antigens can be detected in the blood prior to development of overt symptoms and these have both diagnostic and prognostic clinical value. Newly discovered autoantigens suggests that there may exist additional blood-borne markers of Type 1 diabetes of diagnostic importance.
The current diabetes Type I project seeks to identify novel autoantibodies through serological screening of diabetic blood samples, using an array expressing a 6000+ library of human proteins. Using the recently developed, high density, in situ-expressed, protein microarray technology—termed NAPPA—this leading edge, high-throughput screening will be pursued.
The Piper Center’s diabetes efforts, by making use of a particularly broad set of potential molecular targets, will attempt to a) identify a panel of serum-reactive antigens to which autoantibodies associated with disease can be detected, by means of multiple confirmatory rounds of screening, and b) test the ability of serum-reactive antigens to accurately discriminate between those with autoimmune diabetes and those without. The achievement of these goals would be a significant boon to medical science, enhancing early detection of diabetes and improving opportunities for disease intervention via antigen-specific immunotherapy.
Further, when these studies are complete we hope to better understand the autogenetic origins of Type I diabetes, through an exploration of the expression, localization and interactions of the antigen, as well as changes in antibody responses over time.
The threat of a malicious attack using a lethal biological agent remains a serious societal concern. One potential use of a bioweapon would be the aerosol release of Bacillus anthracis spores, causative agent of anthrax disease. Recent events have demonstrated the potential for significant illness, societal disruption and loss of life following an aerosol release of B. anthracis spores. Unfortunately, disaster prevention has not kept pace with this emergent threat. The current FDA approved anthrax vaccine (AVA) was licensed 38 years ago and requires a six-dose immunization schedule with annual boosters.
Further, the fact that anthrax is primarily a veterinary disease with limited human incidence means that little human efficacy data pertaining to AVA vaccination exists. AVA also has many reported side effects including localized swelling and pain at the injection site; head, joint and muscle aches; malaise; nausea and fever.
The primary immunogen of AVA is Protective Antigen. While Protective Antigen alone offers a degree of anthrax protection, the addition of cell culture filtrate is known to enhance this protection. Using NAPPA technology, we can identify which proteins of B. anthracis account for this enhanced level of protection. If the total number of proteins used to achieve this level of protection can be reduced through this method, many of the observed side effects attributed to AVA vaccination could be eliminated. In addition, supplementation of a higher dose of newly identified immunogens could provide additional protection and longevity to the immune response, perhaps reducing the number of vaccine inoculations.
To date, the Piper Center has produced a sequence-verified collection of B. anthracis protein coding genes that is 96% complete. All of these genes have been successfully transferred into a plasmid vector that is compatible with NAPPA. The resulting slides are being immuno-screened with blood from test subjects including 1 human inhalation anthrax patient, 7 human cutaneous anthrax patients and 4 human AVA vaccines. Additional blood samples from 9 rhesus macaques that have been vaccinated with dilutions of AVA and inhalationally challenged with fully virulent Ames strain spores, have also been immuno-screened.
We find a specific immune response to Protective Antigen control spots, apparent in both human patients and macaques. We are currently in the process of accessing immunogenicity for the screened experimental B. anthracis proteins. The identified antigens we are uncovering will be prime candidates for subsequent protective immunization studies of anthrax.
The human kinome—the body’s collection of protein kinases—contains more than 500 proteins of critical importance for normal cell physiology. Such kinases are also known to play an active role in many diseases. Kinases, when situated on our NAPPA array, display autophosphorylation activity.
To determine if the kinase activity could be affected by inhibitors, the proteins' autophosphorylation activity was also evaluated when the reactions were performed in the presence of global kinase inhibitors such as ADP and Staurosporine. In this case, the observed reduction of the kinase activity suggested that the proteins on the array can be inhibited by general kinase inhibitors, as expected. Ongoing experiments are using kinase NAPPA arrays to study the effect of specific kinase inhibitors (small molecules) on the kinase activity and determine the small molecule selectivity and efficacy among many tested kinases, all performed in a single experiment. Determined IC50s agree well with published results for solution based assays. This method may provide a high throughput rapid approach for evaluating drug selectivity.
Our interest in this research is in identifying and characterizing genes regulating critical events involved in the progression of breast cancer and in understanding the development of resistance to anti-estrogen drug treatment. In previous work, high throughput screens were used to identify genes contributing to various cancer phenotypes including cell migration and acinar morphogenesis. Recently, efforts have concentrated on identifying specific proteins that contribute to drug resistance in breast cancer.
In order to investigate these protein disparities, we have produced several matching drug sensitive and resistant breast cancer cultured cell lines, useful for studying the factors leading to cancer drug resistance. These cells provide an invaluable resource for studying which genes are inappropriately regulated in the drug resistant state. Through this research we have identified a bio-signature, based solely on differences in the resistant and sensitive subcloned cell lines, that can predict overall patient survival in patients treated with the drug tamoxifen.
Using our clone collection of more than 500 human kinases in high throughput unbiased screens, we were able to identify 29 kinases conferring drug resistance. Several of these kinases were also found in the tamoxifen resistance signature.
The picture is complex. Detailed analysis has now shown that one of these kinases both confers resistance on tamoxifen sensitive cells when ectopically expressed and kills resistant cells when knocked down by shRNA. In summary, our results will lead to important new findings detailing specific genes involved in breast development and resistance to hormonal treatment in breast cancer. We hope that defining gene pathways responsible for drug resistance will lead to combined treatments that will more effectively treat resistant cancers and improve patient outcomes.
Each month, innovative research from the Biodesign Institute appears in the world’s premier scientific journals. Many such studies have graced the covers of these journals, showcasing Biodesign research ranging from water remediation to nanometer-scale structures built from DNA.
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