The Impact of Chemistry on Biotechnology - American Chemical Society

Chapter 29

New Diagnostic Tools from Biotechnology Martin Nash

Downloaded by UNIV LAVAL on April 9, 2016 | http://pubs.acs.org Publication Date: January 7, 1988 | doi: 10.1021/bk-1988-0362.ch029

Corporate Development, Synbiotics Corporation, San Diego, CA 92127

Recent years have seen the development and evolution of new tools in the diagnostic field, tools which derive from biotechnology. Generally, these are concentrated in three technology/product clusters. There are others - but these three are particularly notable. In part this is because they are quite different from each other, and in part because they are at quite different stages in their evolution as useful technologies. Specifically, they are monoclonal antibody development, nucleic acid probe development, and biosensor technology. Monoclonal antibodies and nucleic acid (DNA or RNA) probes are emerging as diagnostically approved, commercial products in diagnostic testing markets. Biosensors have not yet reached that stage of development, but their general form can be discerned on the horizon and they are included in this analysis. I shall discuss these technologies and products in the light of industrial expertise rather than scientific expertise; that is I shall compare and contrast the products of these technologies according to the characteristics that are commonly accepted as determinants of success in the diagnostic industry. No matter how marvelous a technology or how intriguing its scientific possibilities, to become a widely utilized and successful diagnostic product it must meet certain tests of utility, and meet them in a way which provides differential advantage over competing products. This chapter explores how these tools of biotechnology fare against each other in a variety of product characteristics. It will look broadly at the areas in which the products of the new technologies will be employed in diagnostics, will assess their advantages (both absolute and relative) and will examine some of the criteria for commercial success which inescapably apply to any diagnostic product. First, a consideration of monoclonal antibodies. The method for obtaining monoclonal antibodies is well known. The intent of this outline is to serve as a general reminder of many of the important

0097-6156/88/0362-0350$06.00/0 © 1988 American Chemical Society

Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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milestones i n producing a monoclonal antibody-based product, and a l s o to remind of the point that many of the steps are not w e l l automated and so remain labor i n t e n s i v e . PRODUCTION OF MAbs Isolate Antigenic Determinant Immunize Mice Harvest Antibody Producing Lymphocytes Harvest Cultured Myeloma C e l l s Mix Fuse Screen Immunoglobulin Producing Hybridomas Inject In Host Mice or Grow In Tissue Culture Harvest Purify From A s c i t e s Attach Reporter Groups The business of harvesting, f o r most laboratories the business of screening, and c e r t a i n l y the feeding, housing, i n j e c t i n g , and f i n a l l y s a c r i f i c i n g of the mice, contain a substantial labor component. Many would be quite surprised to contrast the salary industry must pay to a person who spends the work day s a c r i f i c i n g mice with the salary paid t o post-doctoral workers in l a b o r a t o r i e s . S u f f i c e i t to say that the work of s a c r i f i c i n g mice and harvesting a s c i t e s f l u i d from t h e i r bodies i s not s u f f i c i e n t l y stimulating nor e x c i t i n g t o draw people to perform i t for i t s own sake. At current production q u a n t i t i e s , the body c a v i t y of the mouse remains the production v e s s e l of choice for monoclonal antibodies, however, when new products involving vaccines and therapeutic products come to market, other production schemes w i l l have to evolve with them. The recognition of the f a c t that i t i s inefficient to produce 10, 20, and ultimately hundreds of kilograms of monoclonal antibody i n mice i s what has stimulated the great i n t e r e s t i n hollow f i b e r , fermentation and other large scale mammalian c e l l culture production methods, and i n f a c t what provided much of the excitement behind the a c t i v i t i e s of ccmpanies o f f e r i n g such products. In any case, issues of p r a c t i c a l i t y of various production methods are the substance of another d i s c u s s i o n , and the r e s t of t h i s paper w i l l assess conditions impacting diagnostic products currently being produced and expected to be produced i n the reasonably near future. We f i n d i n turning to the u t i l i z a t i o n of monoclonal antibodies i n the diagnostic t e s t i n g market there i s r e l a t i v e concensus on t h e i r current, and f o r the next few years a t l e a s t , projected f i n a n c i a l activities. Sales a c t u a l l y began i n the early 1980's and were more or l e s s a m i l l i o n d o l l a r s i n 1980. Depending on which survey one reads, sales i n 1985 were i n the v i c i n i t y of $50 m i l l i o n . Projections f o r 1990 are of course more challenging, but among the various market research and forecasting organizations there is rather remarkable agreement that sales of monoclonal antibodybased diagnostics w i l l be i n the $200 m i l l i o n area, with estimates ranging from about $175 m i l l i o n to about $210 m i l l i o n . These figures are worldwide with a b i t over h a l f the t o t a l concentrated


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i n the United States. It i s i n t e r e s t i n g that the same forecasters project that growth of revenues o v e r a l l i n the diagnostic reagent marketplace w i l l be about 8% per year between now and 1990. The projections work out to 15% to 20% per year f o r monoclonal antibody-based d i a g n o s t i c s . If there was any question why companies are aggressive i n MAb product development, these growth data supply the answer. Monoclonal antibodies are primarily employed i n two broad product c l a s s e s ; replacement products and novel products. The e a r l y monoclonal antibodies, as would be expected with any new technology, were l a r g e l y replacements f o r the polyclonal antisera component i n such products as pregnancy t e s t s . Such substitutions c a r r i e d the least technological and market r i s k yet provided more "sizzle with the steak" and often lower cost than their predecessor products. The market has progressed, and the science underlying i t has progressed, to permit the production of antibody s p e c i f i c i t i e s which make i t possible to both replace and even expand product l i n e s . This i s important to industry because i t makes i t possible to avoid competing on p r i c e and begin to release products based on improved performance c h a r a c t e r i s t i c s , which are commonly translated i n t o higher prices than the products they replace. An example of such a development i s the replacement of T - c e l l tests which simply enumerate the number of T - c e l l s i n a patient sample with monoclonal antibodies which make i t p o s s i b l e , and i n f a c t even e a s i e r , to enumerate t o t a l T - c e l l s , t o t a l Bc e l l s , and i n f a c t the r a t i o s of c e r t a i n s i g n i f i c a n t subsets of Tcells. Finally, companies can produce monoclonal antibody products which perform t e s t s which are not replacements for older assays but rather are products uniquely possible as a r e s u l t of the s p e c i f i c i t y of monoclonal antibodies. It i s i n such products that the highest sales value can be derived from the technology; and so these are the most a t t r a c t i v e to companies. Examples of such t e s t s are the tumor marker t e s t s currently emerging i n the cancer market and the t e s t s for natural k i l l e r c e l l s emerging i n c l i n i c a l immunology, and such t e s t s as the Synbiotics' t e s t f o r the heartworm p a r a s i t e . Parasitology has been a troublesome area for antibody companies as parasites commonly pass through d i f f e r e n t l i f e stages where d i f f e r e n t antigens are expressed. Further, parasites are t y p i c a l l y d i f f i c u l t to d i s t i n g u i s h with polyclonal antibodies due to c r o s s - r e a c t i v i t y . The heartworm i s deposited i n the bloodstream of several mammals by mosquitos; it then colonizes the heart muscle i n numbers from 30 to a few hundred. The S y n b i o t i c s assay i s a 20 minute t e s t f o r a shed antigen. 1

There a r e , however, areas of t e s t i n g which provide medically useful information, but which are not amenable to any form of antibody analysis. These include analysis of genetic abnormalities or disease susceptability at the gene l e v e l . Such assays, and others which I w i l l discuss presently, are thought to be the province of DNA and RNA probe technology.


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In turning our attention to probes, f i r s t we should note that not a l l probes are created equal, nor i n f a c t i n the same way. "Long" DNA probes are the more t r a d i t i o n a l e n t i t y , while "short" DNA probes are the more recent development produced s y n t h e t i c a l l y , often on DNA synthesizers currently manufactured by some eight companies. In "long" probe production, DNA from a target of interest (for example, a v i r u s or pathogenic bacterium) i s i s o l a t e d . Ihe broth i s p u r i f i e d to obtain DNA free of d e b r i s , which i s then inserted into a suitable vector (eg plasmid, bacteriophage). These molecules are then cloned thereby r e p l i c a t i n g the inserted DNA and t h i s DNA of interest i s then removed by r e s t r i c t i o n enzymes. The r e s u l t i n g material i s p u r i f i e d (for example on a gel) and then labeled with signal-generating reporter groups (or t h e i r precursors) using polymerases or other appropriate enzymes. B r i e f l y , note that there are a f a i r number of steps, and that many of the steps involve p u r i f i c a t i o n of a s o l u t i o n , steps which are not well l i k e d by manufacturing concerns since they are both expensive and exceptionally troublesome. PRODUCTION OF "LONG" PROBES Isolate DNA Fragment from Target Molecule S e l e c t i v e l y R e s t r i c t , Isolate and Test Target Fragment Insert DNA into a Vector Molecule, e . g . Plasmid Propagate Host Molecule Enzymatically R e s t r i c t DNA Purify Identify and Isolate Desired Probe Fragment Enzymatically Incorporate Reporter Group Isolate Labeled Probe The production cycle for "short" DNA probes i s considerably simpler, and does not involve the complex p u r i f i c a t i o n steps. To produce "short" probes using chemical DNA synthesis, one begins by i d e n t i f y i n g a desired sequence. The chemist must know the sequence desired before he can program the microprocessor i n the synthesizer. It i s c e r t a i n l y worth remembering that this requirement for sequencing the target and i d e n t i f y i n g the p a r t i c u l a r variable region of interest does not e x i s t f o r "long" probes. A single "short" probe synthesis can produce enough material for m i l l i o n s of t e s t s , t y p i c a l l y i n a s i n g l e day. PRODUCTION OF "SHORT" PROBES Obtain Target Sequence Chemically Synthesize Modified Probe Purify Attach Reporter Groups Using nucleic acid probes to detect RNA rather than DNA i n a patient sample i s an i n t e r e s t i n g and i n some ways advantageous approach. Under some conditions, RNA probe duplexes are more stable than DNA probe duplexes, so the assay can be simpler.


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Further, the RNA probes have advantage i n that the user does not have to p r e - t r e a t the specimen to separate strands of double stranded native DNA. This advantage saves time and reduces the technical complexity of the assay. It a l s o means that i n the f i n a l assay the probe need not compete with complimentary strand to anneal to the target strand. RNA probes are commonly intermediate between long and short DNA probes i n length. RNA probes tend to generate somewhat l e s s s i g n a l than either antibodies or "long" probes, but more than "short" probes. It i s not necessary to determine a target sequence p r i o r to producing an RNA probe. There are currently two RNA probes approved f o r diagnostic use, both from GenProbe i n San Diego. The assay employs s o l u t i o n h y b r i d i z a t i o n , thus has substantially shorter performance times than DNA probe assays, and provides more s e n s i t i v i t y than DNA probes because there i s substantially more target RNA than native DNA i n the sample. There are technical problems to be overcome, and RNA probes are not used to detect the presence of viruses since t h e i r current methodology detects ribosomal RNA and viruses lack ribosomal RNA. Nevertheless, t h e i r ease of use, speed, and s e n s i t i v i t y do impart some inherent advantages to RNA probes over DNA probes. Technical and production aspects do not i n themselves determine the d e s i r a b i l i t y of a technology to industry. They must be viewed through the f i l t e r of c h a r a c t e r i s t i c s which apply to a l l i n v i t r o diagnostic t e s t s and determine t h e i r s u i t a b i l i t y f o r development i n t o product. These are not the only elements which enter i n t o a product d e c i s i o n , but they do enter i n t o a l l d e c i s i o n s : BASIS OF COMPETITION Cost of Production Ease of Production Procedural Ease Sensitivity Specificity Technical F a m i l i a r i t y Time of Product Development OOST OF PRODUCTION ο As used i n t h i s context, cost of production w i l l apply to those costs d i r e c t l y a t t r i b u t a b l e to production of the antibody. It does not encompass acquiring c a p i t a l equipment since tax/depreciation impacts vary widely among companies f o r the same plant and equipment. A d d i t i o n a l l y , the product volume against which equipment can be written o f f i s conjecture at present. In the case 'of the MAbs, t h i s term presumes murine production y i e l d i n g some 5 ml of ascites f l u i d per mouse and 5 to 10 mg per ml of antibody.


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EASE OF PRODUCTION ο Ease of production skews the mix of desirable products toward the better known of the technologies i n question (MAbs) and the more automated (short probe). As diagnostic companies are not primarily pursuing new information of a s c i e n t i f i c nature, but rather the most e f f i c i e n t and f a i l u r e - p r o o f production technology, anything which involves a great deal of "art" or extreme complexity w i l l be r e l a t i v e l y disadvantaged. Art particularly i s anathema t o r a t i o n a l production decisions and to the regulatory and supervisory mechanisms i n the healthcare industry of most countries. This acts to the detriment of such products as "long" probes.


PROCEDURAL DIFFICULTY ο This aspect eases t h i s consideration i n t o the area which companies c a l l the marketplace and consumers c a l l the laboratory. The term could a l s o be c a l l e d laboratory procedural ease. It is common f o r t e s t s which are complex to be run l e s s successfully by technicians i n the diagnostic laboratory, who are usually not interested, nor should they be, i n blazing new research t r a i l s or in creating unique diagnostic events. Perhaps the most overwhelming advantage of MAbs over any type of probe assay i s that they are used i n f a m i l i a r assay formats, formats compatible with e x i s t i n g instrumentation. SENSITIVITY ο This i s commonly, i f incompletely, defined as p o s i t i v i t y in the disease s t a t e . Monoclonals have an i n t r i n s i c advantage over the competing probe methodologies discussed herein simply because of the number of copies made of DNA, RNA and proteins respectively. One copy of DNA makes many copies of RNA, which then makes many copies of p r o t e i n . Monoclonal antibodies able to recognize each manifestation of the protein thus have an inherent advantage i n s i g n a l generation. In t h i s respect, MAbs have the advantage of thousands of antibody binding s i t e s per target e n t i t y . RNA probes s i m i l a r l y have more RNA t o "look at" than DNA probes have DNA. SPECIFICITY ο Defined as negativity i n non-disease, t h i s i s a c r i t i c a l but complex i s s u e . Probes are inherently s p e c i f i c f o r the target of interest since they look d i r e c t l y at the DNA, hopefully at the region which codes f o r the i d e n t i t y of the organism i n question or the stretch which defines the target of i n t e r e s t . In the "long" probe embodiment, and i n the RNA probe embodiment, often the target i s not sequenced and thus other regions are included i n the area with which the probe i s r e a c t i v e . This causes most investigators to report that i t i s d i f f i c u l t to detect one or few base p a i r differences between d i f f e r e n t organisms with a "long" probe.



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ο Monoclonals are able t o recognize a target p r o t e i n s i t e c o n s i s t i n g of as few as f i v e amino acids. This i s useful when a s i t e c o n s i s t i n g of f i v e amino acids i s unique t o the target, but can i n f a c t be something of a problem i f the i d e n t i c a l sequence i s present on an unrelated p r o t e i n . The example i s that one of the commonly used monoclonal antibodies t o human T - c e l l s was found a f t e r some two years of wide-spread use t o be present on normal b r a i n t i s s u e . That i s t o say that the high s p e c i f i c i t y imparted by recognizing an antigenic s i t e of as few as f i v e amino acids can lead t o c r o s s - r e a c t i v i t y with the same s i t e i n unrelated molecules or t i s s u e s . This problem i s minimized i n diagnostic assays, p a r t i c u l a r l y of f l u i d s , by r e s t r i c t i n g the a p p l i c a t i o n to that f o r which the t e s t i s shown t o be s p e c i f i c . I t i s , however, not infrequently a complication i n research a p p l i c a t i o n s . Nature has generally been kind to immunologists i n producing markers which permit monoclonals t o detect a l l relevant s t r a i n s but no others. Companies producing monoclonal antibodies must presume that t h i s s i t u a t i o n w i l l hold i n future. TECHNICAL FAMILIARITY ο This r e f e r s to the f a m i l i a r i t y of the customer with a given test protocol. Many e x i s t i n g diagnostic techniques ( f o r example ELISA) are somewhat complex procedures and yet are r o u t i n e l y practiced. Procedural d i f f i c u l t y i s not, therefore, i n i t s e l f a barrier t o the entrance of a product i n t o the diagnostic marketplace. I t i s more procedural f a m i l i a r i t y that can r e t a r d new entrants. C l e a r l y , monoclonal antibodies, and t o a l e s s e r extent RNA probes when used i n s o l u t i o n h y b r i d i z a t i o n t e s t i n g , hold an overwhelming advantage over DNA probes which must be baked onto f i l t e r s or used on d i f f i c u l t - t o - h a n d l e s o l i d state surfaces. TIME OF DEVELOPMENT ο For MAbs t h i s i s defined as the period from i d e n t i f i c a t i o n of the immunogen and i t s p u r i f i c a t i o n t o the release of a product; for "long" probes i t i s the period from a v a i l a b i l i t y of a DNA fragment t o the product; f o r "short" probes i t i s the time from which the target sequence i s known t o the product. Regulatory issues are e x p l i c i t y omitted from t h i s timetable. More generally, DNA probe t e s t s w i l l involve challenging extraction, sample purification, h y b r i d i z a t i o n , and some s u b s t a n t i a l form of )preferrably non-isotopic) a m p l i f i c a t i o n of s i g n a l . As a r e s u l t , basic instrumentation work needs t o be done. This w i l l r e t a r d t e s t development. So the question i s , Where does the advantage l i e i n the "current state of the a r t " ? As we have defined the common bases of competition, the advantages are described as f o l l o w s :


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BASIS OF COMPETITION Cost of Production East of Production Procedural Ease Sensitivity Specificity Technical F a m i l i a r i t y Time of Product Development

ADVANTAGE Short Probes, RNA Probes Short Probes Antibodies Antibodies, Long Probes Roughly Equivalent Antibodies Short Probes, RNA Probes

As i s common with data when presented i n t h i s format, the p a r t i c u l a r c h a r a c t e r i s t i c s are not weighted. When a company weights these various c r i t e r i a , p a r t i c u l a r s i g n i f i c a n c e i s given to the customer s a b i l i t y to u t i l i z e the product successfully (procedural ease, technical f a m i l i a r i t y ) , to a b i l i t y to make the product s u c c e s s f u l l y , and to cost of production. In none of the cases described above i s the cost of production p r o h i b i t i v e for tests carrying the sorts of p r o f i t margins t y p i c a l i n the diagnostic industry, so f o r the purposes of t h i s evaluation cost of production i s roughly the same between "short' probes and RNA probes and i s not r e a l l y s i g n i f i c a n t l y more for antibody production i n the quantities required for a diagnostic assays, so t h i s element i s not c o n t r o l l i n g . Clearly then from the weighted analysis of these bases of competition, the current advantage is with monoclonal antibodies. A factor which explains every b i t as much as t h e i r experience advantage why they are the biotechnology t o o l most commonly employed by companies today.


1

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Considering inherent technology - the two tools d i f f e r as w e l l . On the f a r l e f t are products with c l e a r MAb inherent advantage far right, probe advantage r e s i d e s . This table illustrates product areas where one or another leads.

APPLICATIONS OF MAbs AND NUCLEIC ACID PROBES Tumor Associated Cancer Cancer Antigens Diagnostics Susceptability

Latent Viral

TDM

Immunoassay Replacement

MAb Advantage

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The Impact of Chemistry on Biotechnology - American Chemical Society

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