Multiplex Assays: Evolving Technologies, Applications and Future Directions

Transcription

1 Multiplex Assays: Evolving Technologies, Applications and Future Directions Ken Rubenstein, PhD InsightPharmaReports.com

2 Multiplex Assays: Evolving Technologies, Applications and Future Directions by Ken Rubenstein, PhD Published in January 2010 by Cambridge Healthtech Institute Reproduction prohibited i

3 Multiplex Assays: Evolving Technologies, Applications and Future Directions by Ken Rubenstein, PhD About the Author Ken Rubenstein, PhD, a biochemist and molecular biologist, received his PhD at the University of Wiscon sin and postdoctoral training at the University of Pennsylvania School of Medicine. He was a key innovator and research man ager for Syva Company, the diagnostics branch of Syntex Corporation. During his 13 years with Syva, Dr. Rubenstein became vice president, scientific affairs, a function that included strategic planning. Since 1983, he has served as a technology and marketing consultant to biomedical companies and an industry analyst, with more than 40 published studies to his credit. For more information about published Insight Pharma Reports, visit or call Rose LaRaia at A Cambridge Healthtech Institute publication 2009 by Cambridge Healthtech Institute (CHI). This report cannot be duplicated without prior written permission from CHI. Every effort is made to ensure the accuracy of the information presented in Insight Pharma Reports. Much of this information comes from public sources or directly from company representatives. We do not assume any liability for the accuracy or completeness of this information or for the opinions presented. Cambridge Healthtech Institute, 250 First Ave., Suite 300, Needham, MA Phone: Fax: Reproduction prohibited iii

4 Table of Contents Chapter 1 Introduction... 1 Chapter 2 Evolution of Multiplex Assays and Translational Medicine : The Evolution of Now-Classical Diagnostic Biomarkers... 5 Sensitivity, Specificity, and Predictive Value of Biomarker Assays : Pre-Genomic Biomarkers and Technologies... 7 Enzyme and Metabolite Assays...7 Immunodiagnostics...9 Enzyme Immunoassays...9 Molecular Diagnostics...10 Target Amplification: Polymerase Chain Reaction...11 Signal Amplification Methods : Biomarkers in Drug Discovery and Development : Translational Medicine Chapter 3 Multiplex Technologies : Nucleic Acid-based Multiplex Assays Two-Dimensional Positional Microarrays...19 Affymetrix...19 Agilent Technologies...21 Roche NimbleGen...22 Encoded Particle Arrays...24 Illumina...24 True Materials...25 NanoString...26 Next-Generation Sequencing as Competition for DNA Microarrays.27 Applied Biosystems...28 Roche s NimbleGen...29 Agilent Technologies...30 Others Reproduction prohibited ix

5 Table of Contents 3.2: Multiplex Assays for Proteins Mass Spectrometry...33 Positional Microarrays for Multiplex Protein Analysis...36 Randox Laboratories...36 Aushon Biosystems...36 Theranostics Health...37 Encoded Bead Technology...37 Luminex s xmap Bead Array...37 Bio-Rad...38 Biosite s Triage Assays...38 Other...39 Theranos : Multiplex Assays for Small Molecule Metabolites Metabolon...41 BG Medicine...41 Biosite...41 Chapter 4 Applications : Pharma Eli Lilly...48 Novartis...56 The Theranos Approach : Predictive Toxicology and Multiplex Biomarkers : Diagnostics Ridge Diagnostics...64 Satoris...65 Rules-Based Medicine...65 Proteome Sciences...66 Tethys Bioscience...66 Pathwork Diagnostics...67 BioTheranostics...67 Health Discovery Corporation...67 Quest Diagnostics...68 Celera Corporation...68 Decision Biomarkers...69 Banyan Biomarkers...69 Chapter 5 Market-Related Considerations : The Competitive Environment Deal Patterns User Survey Results x Reproduction prohibited

6 Multiplex Assays: Evolving Technologies, Applications and Future Directions Chapter 6 General Observations and Conclusions Issues Slowing the Deployment of Multiplex Biomarkers Pharma s Shift in R&D Emphasis Translation in Theory versus Practice Translational Medicine-How Well is it Working? Discussion of User Survey Results Chapter 7 Interview Transcripts David Lester, VP, Human Health Solutions, Theranos Brian Edmonds, Ph.D., Research Advisor, Global External R&D, Eli Lilly Stephen Naylor, Ph.D., Founder and Chairman of PPM, Inc Stephen A. Williams, M.D., Chief Medical Officer, SomaLogic Michael Spain, M.D., Chief Medical Officer, Rules-Based Medicine Peter Tolias, Ph.D., Executive Director, Institute of Genomic Medicine, Research Director, The Autism Center, University of Medicine & Dentistry of New Jersey References Company Index with Web Addresses Appendix EXHIBITS Exhibit 3.1 Readout from an Affymetrix DNA microarray experiment Exhibit 3.2 Affymetrix photolithographic process for DNA microarray manufacturing Exhibit 3.3 Roche NimbleGen microarray preparation process Exhibit 3.4 Image of a scan from the NanoString Digital Analyzer showing immobilized and aligned reporter molecules Exhibit 4.1: Consortia and Joint Programs for Developing Multiplex Biomarkers for Predictive Toxicology Exhibit 5.1 Selected Deals Involving Multiplex Biomarkers Exhibit 5.2 Company Category Exhibit 5.4 Work Function/Pipeline Stage Exhibit 5.5 Employment of Multiplex Biomarkers in Preclinical Development Reproduction prohibited xi

7 Table of Contents Exhibit 5.6 Employment of Multiplex Biomarkers in Phase 0 Clinical Studies Exhibit 5.7 Employment of Multiplex Biomarkers in Phase I/IIA Clinical Studies Exhibit 5.8 Employment of Multiplex Biomarkers in Phase IIB/III Clinical Studies Exhibit 5.9 Employment of Multiplex Biomarkers in Phase IV Clinical Studies Exhibit 5.10 Use of the Same Multiplex Biomarkers in both Preclinical and Early Clinical Studies Exhibit 5.11 Would Like to Use Same Multiplex Biomarkers in both Preclinical and Early Clinical Studies Exhibit 5.12 Organizational Preference to Use Single-Analyte Biomarkers Whenever Possible Exhibit 5.13 Reasons to Prefer Single Analyte Biomarkers Exhibit 5.14 Respondent s Organization Uses Multiplex Biomarkers Preclinically without Formal Validation Exhibit 5.15 Respondent s Organization Uses Multiplex Biomarkers in Phase 0 Studies without Formal Validation Exhibit 5.16 Respondent s Organization Uses Multiplex Biomarkers in Phase I/IIA Clinical Studies without Formal Validation Exhibit 5.17 During the Next Three Years, Organization s Expectation for Multiplex Biomarkers in Preclinical Development Exhibit 5.18 During the Next Three Years, Organization s Expectation for Multiplex Biomarkers in Phases I/IIA Clinical Development Exhibit 5.19 During the Next Three Years, Organization s Expectation for Multiplex Biomarkers in Phases IIB/III Clinical Development Exhibit 5.20 Organization has a Drug in Development that Will Have a Companion Diagnostic Exhibit 5.21 Diagnostic in Development is Single or Multianalyte Exhibit 5.22 Organization s View of Companion Diagnostics Exhibit 5.23 Organization Has a Formal Translational Medicine Group or Function? Exhibit 5.24 Contribution of Translational Medicine to Respondent s Organization xii Reproduction prohibited

8 Introduction translational medicine, a field that attempts to facilitate the conversion of basic research findings to medical advances. In pharma, translational medicine means, in large measure, facilitation of early drug candidate attrition, the translation of preclinical proof-of-concept results to human studies, and predictive toxicology. As the biomarker field has developed, for reasons to be explored in this report, the majority of biomarkers that find use in pharma beyond the preclinical development stage are single-analyte varieties, although multiplex biomarkers are known to contribute to early stage human clinical trials. Pharmaceutical companies are also increasingly committing to associating their drugs with diagnostic assays. The majority of these too are single-analyte biomarkers. However a number of multiplex biomarkers not directly associated with particular drugs are in use as approved or homebrew diagnostics, and these contribute in various ways to the broad field of translational medicine. This report examines the role of multiplex and multi-analyte biomarker assays in translational medicine. We deal with their direct contributions to drug discovery and development; their contributions as theranostics, companion diagnostics, etc.; and the reasons why they are not more prevalent despite their apparent high potential. Following this brief introduction, Chapter 2 provides background, definitions, describes the evolution of multiplex assays, and the development of translational medicine as both a concept and a manifest reality. Chapter 3 provides an in depth examination of technological aspects of multiplex assays with emphasis on transcriptomics, proteomics, and metabonomics. The emphasis here is on recent developments and newer players in the multiplex assay field. Also included is a discussion of the potential role of next-generation sequencing in replacing DNA microarray technology for transcriptomics. Chapter 4 examines applications of multiplex assays in translational medicine, and does so from the perspective of pharma R&D, companion diagnostic products, and the new diagnostics as a contributor to translational medicine. Chapter 5 places the emphasis on market perspectives in the field. We examine the competitive environment in omics technology via results of an online survey of people active in the field, and also examine recent deal activity and what it reveals about the market for multiplex assays and their technologies. 2 Reproduction prohibited

9 Applications the diagnostic box and the whole Theranos process. I m glad you pointed that out because that s the way we think too. When you talk to people in pharma, you find they are compartmentalized and you don t get anyone that looks at the patient from a patient perspective. They are all boxed according to their responsibility. You get a biomarker person, a modeling person, a clinical person, and so on. So you re not looking at the individual as a whole. 4.2: Predictive Toxicology and Multiplex Biomarkers Toxicogenomics, toxicoproteomics, and/or toxicometabonomics for predictive toxicology have been under investigation for at least a decade. One or more of them always seems to be just around the corner, but tends to recede into the distance on close inspection. One key objective involves finding multiplex biomarkers that will predict a compound s safety in animal models. Unfortunately, biomarkers from animal studies do not always predict toxicity in humans. In the early days of toxomics, investigations were mainly conducted by individual companies. Over the years, however, it has become apparent that the task is too large for any one company, and pre-commercial consortia have sprung up in response. A number of these were tabulated by Merck Serono researcher Phil Hewitt in a presentation at Cambridge Healthtech Institute s May 2009 Biomarker World Congress conference. 17 He pointed out that most classical biomarkers reflect late-stage events, whereas it is desired for translation to humans to find early evidence of adverse events, particularly with regard to liver and kidney toxicity (see the Appendix for the link to slides from this presentation). In July 2008, EMEA (European Medicines Agency) issued a report indicating that pending further progress, a multiplex biomarker panel consisting of kidney injury molecule-1 (KIM-1), albumin, total protein, beta-2-microglobulin, urinary clusterin, urinary trefoil factor 3, and urinary cystatin C are considered acceptable in the context of non-clinical drug development for prediction of acute drug-induced nephrotoxicity. 18 The study made use of Rules-Based Medicine s muliplex protein assays, and contributory studies were conducted at Merck, Novartis, and the FDA. The foregoing study was initiated under the auspices of the Predictive Safety Testing Consortium, a public-private partnership led by the non Reproduction prohibited

10 Applications 4.1: Pharma Essentially all major pharmaceutical companies have translational medicine functions or the equivalent built into their organizational structures. Companies range from paying little more than lip service to the latest fad for image reasons, through modest efforts to provide focus on the transition between preclinical and clinical development, to significant shifts in structure of the R&D pipeline. Pfizer established a massive biomarker infrastructure in the early 2000s, only to shed a number of its key players and decentralize the effort during the last couple of years. Merck has also shown signs of pulling back on biomarker-centered efforts with the loss of key players, Stephen Friend and Eric Schadt, who came from Merck subsidiary, Rosetta Inpharmatics, which has now been divested. Eli Lilly and Novartis, on the other hand, have both built key aspects of translational medicine into their core R&D strategies. Eli Lilly Lilly has a distinctive approach to the role of biomarkers and diagnostics in pharma. In a May 2009 presentation at Cambridge Healthtech Institute s Biomarker World Congress, John Bloom, Executive Director Diagnostic and Experimental Medicine, revealed that an overarching priority at Lilly is to use diagnostics to optimize the value of the company s drugs in the marketplace (see the Appendix for the link to slides from this presentation). Lilly considers it crucial to its corporate strategy to improve outcomes for individual patients. Bloom is the Executive Sponsor of the Lilly Diagnostic Core Team and Brian Edmonds (see interview in Chapter 7) is the Chair, Global External Research & Development. Team members represent Diagnostic and Experimental Medicine, Global Regulatory Affairs, Law, and Global Business Development. Lilly views the stand-alone business value of diagnostics to be limited compared to their value in the context of new drugs, and they recognize that associated technology and commercialization of diagnostic assays requires multiple, and often novel, partnerships. Examples of diagnostic opportunities recognized by Lilly cover a spectrum from disease markers to target-related markers. Drug portfolio needs drive internal investment and partnership development strategies at Lilly. Depending on needs, varying diagnostic technologies and approaches are employed. These include: 48 Reproduction prohibited

11 Multiplex Technologies An example of a Roche NimbleGen product, the HD2 microarray for Comparative Genomic Hybridization and ChIP-chip analysis contains 2.1 million probe features, each 50 to 75 bases in length, with a 13 micron feature size on a 6.2 x 1.4 cm array. Increased chemical yield per chain elongation step permits NimbleGen to synthesize probes that are considerably long than those provided by Affymetrix. For gene expression, NimbleGen offers chips with one (385K features), four (72K features per arrays), and 12 (135K features chips) arrays. The latter two permit assaying multiple samples in a single run. Encoded Particle Arrays Encoded particle arrays, once a distant second in the marketplace to positional microarrays, have gained in popularity and market share largely through the efforts of two companies: Illumina (San Diego, CA) and Luminex (Austin, TX). In fact Illumina is now the market leader in microarray-based products. These arrays fulfill the same functions as positional arrays, while providing flexible scalability (from lowlevel to massive parallelism) to cover both discovery and repetitive assay applications. Since the particle-based entities are not arrays in the conventional sense, they are sometimes called virtual arrays. In an encoded particle array, each particle contains a capture molecule and an identification code. Capture entities are typically oligonucleotides or antibodies, and codes can be other oligonucleotides or dyes. Luminex technology is discussed in Section 3.2. Illumina Illumina s long-established BeadArray product line is based on 3-micron silica beads that self-assemble in microwells on either of two substrates: fiber optic bundles or planar silica slides. When randomly assembled on one of these two substrates, the beads have a uniform spacing of about 5.7 microns. Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that act as the capture sequences for a particular assay. Illumina s iscan instrument runs BeadArray assays covering a broad range of DNA and RNA analysis applications. Although it is scalable to an extent, BeadArray technology is applicable mainly to high levels of multiplexing. In 2007, Illumina commercialized a second encoded particle technology called VeraCode, which is based on digital holographic encoding. The particles are essentially barcodes imprinted on micron glass microbeads. When excited by a laser, each bead emits its unique code image, which is read using Illumina s BeadXpress 2-color laser detection system. Depending on desired multiplex levels, assays are created by pooling microbeads with code 24 Reproduction prohibited

12 General Observations and Conclusions The now-classical method of comparing expression of a great many genes, proteins, or metabolites in two or more conditions, selecting differentially expressed genes, and conducting preliminary retrospective validation studies on chosen panels has had surprisingly limited success in generating reliable assays for use in Phase III clinical trials or as routine diagnostics. As a general observation, it seems fair to say that this prospecting approach is not likely to yield such assays. Rather, there is a growing sense that biomarkers must be chosen with some rational hypothesis in mind, and indeed a growing number of academic researchers and even some commercial ones are moving their biomarker work in that direction. 6.1 Issues Slowing the Deployment of Multiplex Biomarkers When asked why, despite their potential for increased information content, multiplex biomarkers aren t finding greater use in pharma, David Lester replied: If I look at the clinical level and the effect of personalized medicine, I think a lot of the shift has come because of regulatory impacts. The [FDA] Office of In Vitro Diagnostics is not clear on how to deal with these multivariate or multiplex biomarkers. You ve got a double whammy. First there s a series of individual assays that you have to deal with, and then with a lot of the multiplex assays, you ve got an algorithm as well, which is giving a signature. So I think what you re seeing is that it s a lot more complicated dealing with multiplex assays than with single analytes. There are a lot more issues, which is ironic because medicine is really practiced using multiplex type endpoints. There s sort of a disconnect going on. The value you got from multiplex assays was difficult to figure out, so it was easier just to go with single endpoints. I think it s a trend, and I think the multiplex assays will come back ultimately, because you re really not going to get very much out of a single analyte. In addition to regulatory concerns, pharma must also consider the time and expense required for assay validation. When Lester was asked whether pharma will eventually be forced to do the validation studies required to use them in clinical trials, he responded: 94 Reproduction prohibited