James R. Prudent

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive Detection of Respiratory Viruses

ABSTRACT Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.

Evaluation of a Multiplexed PCR Assay for Detection of Respiratory Viral Pathogens in a Public Health Laboratory Setting

ABSTRACT There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered “true positives,” the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.

Exploiting the Enzymatic Recognition of an Unnatural Base Pair to Develop a Universal Genetic Analysis System

BACKGROUND With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. METHODS We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses. RESULTS RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls). CONCLUSION The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.

Quantifying mixed populations of drug-resistant human immunodeficiency virus type 1.

ABSTRACT In order to survive prolonged treatment with antiretroviral nucleoside analogs, the human immunodeficiency virus type 1 (HIV-1) is selectively forced to acquire mutations in the reverse transcriptase (RT) gene. Some of these mutations are more common than others and have become markers for antiretroviral resistance. For the early detection of these markers, a novel MultiCode-RTx one-step testing system to rapidly and simultaneously characterize mixtures of HIV-1 targets was designed. For cDNA, nucleotide polymorphisms for codon M184V (ATG to GTG) and K65R (AAA to AGA) could be differentiated and quantified even when the population mixture varied as much as 1 to 10,000. Standard mixed-population curves using 1 to 100% of the mutant or wild type generated over 4 logs of total viral particle input did not affect the overall curves, making the method robust. The system was also applied to a small set of samples extracted from infected individuals on nucleoside reverse transcriptase inhibitor therapy. Of 13 samples tested, all were positive for HIV and 10 of the 13 genotypes determined were concordant with the line probe assay. MultiCode-RTx could be applied to other drug-selected mutations in the viral genome or for applications where single-base changes in DNA or RNA occur at frequencies reaching 0.01% to 1%, respectively.

MultiCode-RTx Real-Time PCR System for Detection of Subpopulations of K65R Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant Viruses in Clinical Samples

ABSTRACT We report a real-time PCR assay capable of detecting drug-resistant human immunodeficiency virus type 1 reverse transcriptase K65R mutant virus at a level of 0.5% in polymorphic patient plasma specimens. Fifty-three treatment-naïve and 20 treatment-experienced specimens were successfully genotyped with the new method. Results were in agreement with population sequencing and the labor-intensive single-genome sequencing method.

Extracellular Antibody Drug Conjugates Exploiting the Proximity of Two Proteins

The human Na+/K+-ATPase (NKA) is a plasma membrane ion pump that uses ATP to help maintain the resting potential of all human cells. Inhibition of the NKA leads to cell swelling and death. The results of this investigation show that on cancer cells, the NKA either comes in close proximity to, associate with or complexes to important cancer-related proteins, and thus can be targeted with a new type of precision therapy called the extracellular drug conjugate or EDC. The EDCs reported here exhibit EC50 values in the low to mid-picomolar range, and signal to noise ratios > 1,000:1, both of which are dependent on the cell surface expression of the NKA and corresponding cancer-related target. We demonstrate that a potent small molecule inhibitor of the NKA can be covalently attached to antibodies targeting CD20, CD38, CD56, CD147, or dysadherin, to create a series of selective and powerful EDCs that kill cancer cells extracellularly by a mechanism resembling necrosis. This is therefore a framework for the development of a new type of precision therapy wherein exquisite selectivity is achieved for targeting extracellular disease-related proteins.

Abstract 1202: Target identification for a new type of ADC

Purpose: To explore the mechanism of action of a potent new type of antibody drug conjugate (ADC) called Extracellular Drug Conjugates or EDCs. Experimental Design: EDCs are a new type of ADC that have similar activity and components, yet EDCs are different in the following ways; 1) EDCs do require internalization, 2) EDCs are not pro-drugs but instead require the antibody and payload to remain permanently linked and 3) EDCs target two proteins in unison, requiring long non-cleavable linkers to span distances between bound antibody to the payload binding site. To define the MOA of the EDCs, high definition phase contrast imaging and protein marker experiments were conducted. In order to verify the targets of a set of EDCs consisting of Na,K-ATPase specific payloads, siRNA knockdown experiments were designed and tested. Results: First, we observed that after 24 hour exposure to each of the EDCs (or the free payload), cells rounded up, partially detached from the substrate, swelled, and lost plasma membrane integrity - morphology resembling necrosis. These observations were consistent with no induction apoptosis or autophagy. Second, our siRNA studies determined that alpha 1 of the Na,K-ATPase was a lethal target. Consistent with this observation on all tested cells lines tested, the EC50 values of each EDC decreased with decrease in alpha 1 expression. Additionally, when the corresponding antibody target expression decreased, overall activity of each EDC went down. Alpha 1 expression was also found to change the effects of the free payload, indicating that the EDCs and the free payload share the same target. Yet unlike the EDCs, payload alone was not affected by the expression of any antibody target used in the study. We also observed that siRNA knockdown of the Na,K-ATPase beta 1 or beta 3 subunits alone did not affect EDC or payload activity, yet a significant decrease in activity could be observed when both subunits were knocked down simultaneously. These results are also consistent with microarray data analysis using siRNAs against all 8 alpha and beta subunits which show that only alpha-1, beta-1, and beta-3 are expressed in the cell types tested. Conclusions: These results show that when payloads targeting the Na,K-ATPase are attached to certain antibodies via long flexible linkers, the resulting EDC activity specifically target the alpha 1 subunit of the Na,K-ATPase, of which we determined to be a lethal target. These results also show that EDC9s activity is dependent on the expression of the corresponding antibody target. In addition, the mechanisms of EDCs are similar to that of the free payload and appear to be necrosis like. These results support the continuing efforts to identify the detailed mechanism of the new ADCs which may lead to identifying patients most likely to respond to EDC based therapies. Citation Format: James R. Prudent, Chad Hall, David J. Marshall, Scott Harried, John Murphy. Target identification for a new type of ADC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1202.

Abstract 2964: An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies

Purpose: CD20 is known to be a therapeutic antibody drug target as it is expressed on the surface of most B-cell neoplasms. Here, we assessed the anti-tumor activity of EDC9, a novel extracellular drug conjugate composed of Rituximab (a well know and FDA approved anti-CD20 monoclonal antibody) conjugated to a steroidal glycoside via a long, flexible and stable linker. Experimental Design: The anti-cancer activity and safety of EDC9 were examined and compared to Rituximab using in vitro and in vivo techniques. Results: We found that EDC9 showed picomolar cytotoxic activity that was independent of effector functions and exhibited cytotoxic activity through a mechanism that was dependent on CD20 expression, as cells not expressing CD20 were resistant and Rituximab alone could compete with EDC9, rendering it inactive. After 72 hours of EDC9 treatment at levels between 100 and 200 picomolar, no cells were determined to be viable by CellTiter-Glo or high definition phase contrast imaging. Importantly, when compared on the basis of toxicity to PBMC, EDC9 was found not to be more toxic than Rituximab and the activity of EDC9 was dependent on specific steroid chemistry. In a tumor xenograft model, EDC9 provided complete long-term remission of diffuse large B-cell human lymphoma line SU-DHL-8 tumors, while Rituximab alone did not. Conclusion: These results support efforts to further evaluate EDC9 (a novel CD20 specific antibody drug conjugate) and may lead to phase I clinical trials in patients with certain B-cell related malignancies. Citation Format: James R. Prudent, Chad A. Hall, David J. Marshall, John Murphy, Scott Harried. An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2964.

Abstract A125: Antibody targeted steroids for the treatment of cancer

Purpose: The cardiac glycoside family of steroidal drugs has been shown to possess novel and potent antitumor activities in rodents. Yet most likely due to their cardiotoxic nature, these drugs have failed to show clinical efficacy at doses deemed safe to humans. In order to determine if this drug class could be made safer and more efficacious, we assessed the antitumor activity of a new antibody drug conjugate system in which these steroidal compounds are tethered to antibodies that direct them to protein complexes found on various types of cancer cells. Experimental design: The activity and safety profiles of these novel antibody drug conjugates were examined using multiple cancer cell lines, normal cells, xenograft models in immunodeficient mice and non-human primates. Results: We identified multiple monoclonal antibodies that when conjugated to cardiac glycosides through long stable linkers, could provide cell killing activity independent of effector functions. When tested in vitro on a number of cancer cell lines, these active antibody drug conjugates termed Extracellular Drug Conjugates or EDCs, displayed potent cytotoxic activities with half maximal effective concentration (EC50) in sub-nanomolar ranges via a pathway resembling apoptosis and/or necroptosis. These activities were dependent on the expression of the corresponding antibody, linker length and steroid chemistry. In vivo using various tumor xenograft models, the EDCs were able to partially or completely regress tumor growth when 1 to 10 mgs/kg were administered intravenously. The EDCs were also found to assist various clinically approved therapies regress tumor growth. Finally, because cynomolgus monkeys respond to cardiac glycosides is similar to humans, 5mgs/kg of EDC was slowly infused intravenously into a cynomolgus monkey with no adverse effects observed even though the relative blood concentration of the cardiac glycoside attached reached 1500 nanomolar. Conclusion: These results support efforts to further evaluate these unique targeting antibody drug conjugates for the treatment of human cancers. Citation Format: James R. Prudent, David Marshall, John Murphy, Chad Hall, Scott Harried. Antibody targeted steroids for the treatment of cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A125.