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Biotecir~oZo~ for Healed Care J. PAUL BURNETT Biotechnology has been an applied science in the pharmaceutical in- dustry for a long time. The antibiotics that are used so extensively today in clinical medicine are products of fermentation or biotechnology. These substances have been produced on a very large scale for the last 30 or 40 years. Until a few years ago, however, the organisms used in bio- technology within the pharmaceutical industry were all isolated from nature. Existing organisms were selected using screening procedures designed to detect organisms producing useful substances. Today the tools of biotechnology have changed. Molecular biologists have provided ways of designing and manipulating organisms to produce substances in which there is specific interest, rather than simply accepting what nature has already provided. This advance primarily accounts for the recent excitement within the pharmaceutical industry insofar as bio- technology is concerned. In the pharmaceutical industry biotechnology generally encompasses two primary areas. The first is immunology, in which the discovery of hybridomas and cell-fusion technology have allowed the production of monoclonal antibodies. This has already led to some very important new diagnostic techniques, and it offers the promise of therapeutic ap- plications in the future, but so far none of the latter has really been reduced to practice. This discussion focuses on the second area of bio- technology or biology that forms an important segment of the new technology in the pharmaceutical industry, namely, the area of recom- binant DNA (deoxyribonucleic acid), where many discoveries have al- 62

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BIOTECHNOLOGY FOR HEALTH CARE 63 ready been reduced to practice and where at least one product is already on the market. The concept of recombinant DNA is based on the relatively well- understood function of DNA within all cells. A brief review of some basic concepts of molecular biology can help clarify the ways in which molecular biology and biotechnology can be used. Figure 1 shows the management information system of all cells. All cells contain genetic information stored in DNA. This information is transcribed from the DNA into a working pattern called messenger RNA (ribonucleic acid). This pattern is used in the cell to produce proteins. Proteins are the molecules in the cell that give rise to all of the characteristics that are recognized for particular cells. Some of these protein molecules are enzymes, or biocatalysts, that catalyze the formation of all of the other molecules in the cell. Some DNA (Genetic Information) Messenger RNA Proteins -Transcription -Translation Enzymes Structural Proteins Biological Messengers 1. Low-molecular-weight metabol ites a. Sugars, fatty acids, amino acids, alcohols, etc. b. Antibiotics, antifungals 2. Macromolecules 1. Physical structure 1. Hormones 2. Carrier molecules 2. Mediators FIGURE 1 Management information system of all cells. DNA controls the synthesis of cellular proteins, which subsequently determine the phenotypic characteristics of the cell.

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64 NEW FRONTIERS IN BIOTECHNOLOGY - / \ Plasmid DNA / Protein \ ~ ~Ribosomes i0 / - \ DNA In\ ib / / 00 Protein l (~:~ ;y / / ~ / \ `, imRNA / / mRNA~/ / \ / ~ ''<~ iRibosomes ~ ~ it' / ~ I) ~Chromosomal DNA Animal Cell Bacterial Cell FIGURE 2 Pictorial presentation of management information of cells. In addition to chromosomal DNA, bacterial cells often contain small, autonomously replicating DNA molecules called plasmids. of the proteins serve a structural function, giving rise to the physical appearance in the structure of the cell. Others serve messenger func- tions, carrying messages back and forth between the cells. The sum and substance of a cell is the complement of proteins that it contains. Figure 2 presents much the same information as that in Figure 1, but in pictorial form, and it introduces an additional concept. On the left- hand side of the figure is a typical animal cell where DNA is in the form of chromosomes in the nucleus. The messenger RNA is made in the nucleus and goes out to the cytoplasm where it serves as a pattern for protein synthesis. The same process occurs in bacterial cells (right-hand side of Figure 2) except that bacterial cells have small DNA molecules (known as plasmids) that have particular advantages for use in biotech- nology. As opposed to the chromosomal DNA, which is a very large and very fragile molecule, the plasmid DNAs are very small molecules. They can be isolated in a test tube. They can be cut apart and put back together. DNA can be added to them or subtracted from them. And, finally, plasmids can be put back into a cell in a functional form. Thus, plasmids form the cornerstone, if you will, of the application of molec- ular biology to biotechnology and recombinant DNA.

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BIOTECHNOLOGY FOR HEALTH CARE BIOTECHNOLOGY IN THE PHARMACEUTICAL INDUSTRY 65 Figure 3 indicates how, in a general way, one might use recombinant DNA to produce a substance via biotechnology in the pharmaceutical industry. In the upper left of the figure is a recombinant DNA organism being made by combining an animal gene with a plasmid DNA, followed by introduction into a microorganism. This first step, introducing the recombinant DNA into the microorganism, is a laboratory process. In the laboratory one would generate test-tube-scale cultures that contain the transformed cells that will now produce the protein coded by this animal gene. The next stages are development and production processes. This culture must be scaled up from the test-tube stage to the bioreactor, or fermenter, stage. The product must then be purified and packaged in suitable clinical form. Finally, before the product is ever subjected to clinical use, it is extensively tested in animal systems. Is is important to point out that the bulk of this overall process begins CUT PLASMID Q ANIMAL <=3 ~ GENE PURIFICATION J RECOMBINANT DNA PASSAGE _ THROUGH ADSORBING COLUMNS _ PACKAGING GROWTH IN LARGE TANKS . 0 0 0 0 INSERTION LABORATORY INTO BACTERIA TESTS me) ' 1(~) / ~ :~; 5~ 3 1 THERAPEUTIC VALUE TESTED IN ANIMALS ~ ~- CLINICAL USE FIGURE 3 Production of pharmaceuticals by recombinant DNA. Recombinant DNA can be used to add new genes to microorganisms, and these can be grown in fermentation tanks to produce proteins on a large scale. Purification and extensive testing in animals precede clinical application in human beings.

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66 NEW FRONTIERS IN BIOTECHNOLOGY TABLE 1 Amino Acid Residues and Molecular Weight of Human Polypeptides Potentially Attractive for Biosynthesis Polypeptide Amino Acid Molecular Residues Weight Insulin 51 5,734 Proinsulin 82 Growth hormone 191 22,005 Calcitonin 32 3,421 Glucagon 29 3,483 Corticotropin (ACTH) 39 4,567 Prolactin 198 Placental lactogen 192 Parathyroid hormone 84 9,562 Nerve growth factor 118 13,000 Epidermal growth factor 6,100 Insulinlike growth factors (IGF-1 and IGF-2) 70, 67 7,649, 7,471 Thymopoietin 49 after the genetic engineer has completed his or her work. In a sense, the contribution of the molecular biologist, although crucial, is a small portion of the total process. Using the general process just described, a number of different types of molecules that have potential therapeutic use can now be made. The general categories of these substances include hormones and growth factors, pain-relieving proteins, plasma proteins, enzymes, proteins in the immunology area, and possibly even new types of antibiotics. Table 1 lists some of the growth factors and hormones that one might consider producing by this technology. The genes for almost all of these proteins have now been cloned, and it is possible today to use those genes to produce these proteins in microorganisms. One at least, human insulin, has now been produced on a large scale and is a marketed product. It will be useful here to illustrate how genetic engineering is actually used to produce human insulin. Production of Human Insulin Theoretically there are two ways in which one could go about pro- ducing the insulin molecule (see Figure 4~. Insulin consists of two dif- ferent protein chains, the so-called A chain and the so-called B chain. One could produce the normal precursor of insulin, proinsulin, that is

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68 NEW FRONTIERS IN BIOTECHNOLOGY FIGURE 5 Schematic presentation of process for producing biosynthetic human insulin. Strains of E. cold have been engineered to produce insulin A and B chains. The initial cellular protein product is a chimeric protein in which the insulin polypeptide chain is attached at the carboxyl terminus of a protein coded by the tryptophan operon. Cleavage by cyanogen bromide releases the insulin chain from the chimera. found in the pancreas. Proinsulin is a molecule that contains insulin but also contains an extra connecting peptide linking the two chains together. In the pancreas gland, this so-called connecting peptide is clipped out, leading to the production of insulin that is then released into the blood circulation system. One can mimic this process today in the laboratory and actually even in production, but up to the present it is not being used to produce human insulin on a large scale. ~ .. . . _ . . Presently the A chain and the B chain are made individually, and then these are coupled in the plant to produce the bioactive insulin. Figure 5 shows the process schematically. In separate plasmids the genes have been introduced individually for the A chain and B chain of insulin, and then these plasmids have been

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BIOTECHNOLOGY FOR HEALTH CARE 69 transformed into bacterial cells. The Escherichia cold are then grown in large fermentation facilities. The product that is initially made is a large chimeric protein consisting of the A chain or B chain attached to the end of a naturally occurring E. cold protein. This protein is subjected to a cleavage reaction in which the A chain and the B chain are chemically cleaved away from the rest of the chimeric molecule. Then, following several further purification steps, these two chains are combined, and the biosynthetic human insulin is recovered and purified. Large amounts of the gene product of this plasmid accumulate in the E. coli. A thin-section electron micrograph of E. cold producing human insulin polypeptide shows dense areas, which are deposits of that protein within the cell. The protein is produced in very substantial amounts and can occupy a major portion of the cell. This is one of the advantages of biotechnology today by using ap- propriate control systems and regulatory systems on the plasmid being dealt with, one can make the protein of interest a major portion of the total protein of the microorganism. It can become a very efficient proc- ess. Figure 6 shows crystals of the final product. It is a crystalline protein, F~GuRE 6 Crystals of biosynthetic human insulin produced by the process described in Figure 5.

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70 NEW FRONTIERS IN BIOTECHNOLOGY and it has all of the characteristics of the insulin that is circulating in all of our bodies. There are at least two advantages to being able to produce human insulin as opposed to continuing to use the pork and beef insulin that is currently used in many diabetic patients. First, the chemical structure of pork and beef insulin differs slightly from that of human insulin. Thus, there is the possibility of an improved therapy by using a molecule identical to the insulin that is already circulating in human bodies. The second advantage relates to the fact that currently produced pork and beef insulins are really by-products of the meat industry. Their produc- tion is subject to all of the economic pressures of the meat industry in terms of supply of pancreas glands. By production in microorganisms an essentially limitless supply of human insulin is available; the supply is no longer subject to the particular economic pressures of the beef and pork markets. The following are some of the plasma proteins that one might consider producing by this technology: Albumin Globulins a,,B,~y Lipoproteins a, ,B Plasminogen Fibnnogen Prothrombin Transfernn Albumin, for instance, is a protein that can now be manufactured using recombinant-DNA technology. At least one company is working to scale this process up to commercial levels. Many of the genes for other pro- teins in the plasma protein series have also been cloned. Other Uses of Recombinant DNA Following is a brief discussion of examples of other ways in which recombinant DNA could be used to make products useful in the phar- maceutical industry. Table 2 lists enzymes that are now used clinically. Probably the most important group today is that of the enzymes and cofactors involved in hemophilia proteins like Factor VIIIc, which is used in hemophilia A. A large number of research groups are trying very hard to clone the gene for this protein; although the goal has not yet been reached, it can be expected that this will be accomplished in the near future. One area of particular excitement is the fibrinolytic area. Blood- clotting problems, thrombosis, are an important aspect of clinical med-

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BIOTECHNOLOGY FOR HEALTH CARE TABLE 2 Ethical (Prescription) Enzymatic Products Currently Employed in the United States 71 Enzymes Therapeutic Category Involved Indications Approximate Mfrs. Sales (millions of dollars) Blood clotting factors Antihemophilic Factor Factor VIII Hemophilia A (AHF) Plasma thromboplastin Factor IX Hemophilia B component Gastrointestinal digestive Pepsin, pancreatin "Nervous" or other indigestion Pancreatin: lipase, Inadequate fat digestion; trypsin, amylase cystic fibrosis Wound debriding agents Fibrinolysin and deoxyr~bo- nuclease Trypsin Subtilains Collagenase ~ <1 Streptokinase and Clot lysis, reduction of 1 streptodornase edema and inflammation Chymotrypsin "Possibly effective" for Bromela~ns . . Papain ep~s~otomy Streptokinase Urokinase Fib ri no lysin Hyaluronidase Chymotrypsin Asparaginase Penicillinase Oral proteolytic preparations Thrombolytic Absorption promoter Ophthalmic surgery Cancer chemotherapy Allergic drug reaction 40 4 11 4 ( Removal of purulent exudates and eschar 2 2 3 | Lysis of intravascular <2 blood clots Rarely for IM or SC in Cataract removal Leukemia Destroy penicillin 1j. <1 <1 <1 <1 icine. A number of enzymes, such as streptokinase or urokinase, will degrade blood clots. The problem with both of these enzymes is that although they hydrolyze fibrin and destroy clots, they also lead to gen- eralized bleeding in the patient. A new protein has recently been dis- covered, called the tissue activator of plasminogen (TPA), which is very specific and will hydrolyze fibrin only when it is in the form of a clot. This enzyme does not cause the side effect of generalized bleeding. The gene for TPA has been cloned, and one can now produce this protein using recombinant-DNA technology. One of the most exciting areas for the application of biotechnology within the pharmaceutical industry is in immunology, where it will be

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72 NEW FRONTIERS IN BIOTECHNOLOGY _ Factors Affecting Inflammation Helper Factors May Be Suppressor Factors May May Be Useful in Useful in Be Useful in Patients with overwhelming Tumor patients Allergy infection Postsurgical immune Aging Autoimmune disease, suppression SLE, arthritis Burn patients Diabetics M.S., thyroiditis, myasthenia gravis, etc. Postsurgical peritonitis Dialysis patients Transplantation Tumor patients Immunodeficiency disease Aging Al lergy Immunodeficiency disease NK cell activity Dialysis patients Burn patients Thyroiditis Trauma M.S.-EAE Postsurgical immune suppression Allergy Chronic diseases hepatitis, parasitic Hodgkin's disease FIGURE 7 Clinical applications of cytokines. possible to produce some interesting vaccines. Instead of discussing these possibilities, however, let us turn to a group of proteins called cytokines. A cytokine is a molecule made in a cell as a result of some sort of stimulus. It can result from various types of stimuli. A cytokine is elab- orated by the cell producing it. It leaves that cell and acts as a messenger, one of the functions of proteins mentioned earlier. It then stimulates a second type of cell to cause some sort of biological effect. It may affect the growth of the cell, it may affect the movement of the cell, or it may activate the cell to perform a specific function. For instance, in the phagocytic series it may activate a cell to become more active phago- cytically. Figure 7 illustrates some of the areas where cytokines might be used clinically. Many cytokines are involved in the inflammatory response. Others promote the immune response, and still others are known to suppress the immune response. It is thought that the clinical states shown in Figure 7 represent those in which many of these cytokines might be used therapeutically. The important point with regard to this figure is that it represents a very large number of clinical diseases. Figure 8 shows some of the cytokines that are produced by one par- ticular type of human-cell, the lymphocyte. It can be seen, for instance,

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BIOTECHNOLOGY FOR HEALTH CARE Mediators Affecti ng Macrophages Migration Inhibitory Factor (M I F) Macrophage Activating Factor (MAP} Chemotactic Factor {CF) Mediators Affecting Lymphocytes Allogenic Effect Factor {AEF)Katz Mitogenic Factor {MF) _ _ . . . .. . Factors Enhancing Antibody Formation Factors Suppressi ng Anti body Formation T-Cell Replacing Factor (TRF} Chemotactic Factors for Basoph i Is ~ BC F and Eosinophils (ECF) Mediators Affecting Other Cells Cytotoxic Factor (Lymphotoxin) Cal lagen-Produci ng Factor OsteoclastActivating Factor I nterferon ~ I F ~ FIGURE 8 Cytokines produced by lymphocytes. 73 that there are mediators made that affect macrophages, the cells that destroy invading organisms in the body. There are mediators that affect other lymphocytes and cause them to do a variety of things. Some may enhance, for instance, antibody formation. There are Chemotactic fac- tors elaborated by the lymphocyte that affect cell movement. One of the cytokines that has been most publicized in the recent past is inter- feron, which is made by the lymphocyte, among other types of cells. All of the examples just examined illustrate the type of products that can reasonably be expected to be produced by biotechnology in the pharmaceutical industry. However, exciting as these present applications for recombinant DNA may be, it appears that the total number of drugs that will be produced by this technology as proteins will be limited. In the future the greatest value of the application of biotechnology and recombinant DNA within the pharmaceutical industry will probably come about as we begin to understand life at the molecular level. In addition to offering a way of producing molecules, recombinant DNA offers the molecular biologist a way of cloning and isolating genes, characterizing these genes, and understanding their function at a genetic level. In fact, today it is sometimes much easier to isolate the gene for a particular protein and to learn the structure of the protein from the gene than it is to isolate the protein itself.

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74 NEW FRONTIERS IN BIOTECHNOLOGY All of the processes of life, and especially those orderly processes such as differentiation and development seen in the nodal growth of plants and animals, are ultimately controlled by DNA. This is also true of the disorderly processes that we recognize, for instance, malignant cell growth. As we define these life processes at a genetic level, it is expected that it will be possible to design new small molecules that may be produced by traditional chemical means which will be the drugs of the future; but the discovery of these drugs will hinge on the application of molecular biology. Following is one example of how that might happen and of one area where this might be applied. Genes known as oncogenes have been discovered; they have been found in a variety of tumor viruses from various types of animals. It is also known that there are corresponding genes in normal cells that seem to be very similar in structure to the oncogene of the tumor virus. When a tumor virus invades a cell, the function of the oncogene is to produce a protein that leads then to the transformed or malignant phenotype. The function of the corresponding gene in the normal cell is to regulate the growth and function of the cell. We believe that it will be possible in the future, by understanding how these genes are genetically regulated and how these proteins func- tion, to develop small molecular inhibitors that may be intriguing new types of chemotherapy for treating cancer. It is certainly too early to say what form these will take or exactly what structure they might be, but it is an interesting example at least of a way in which recombinant DNA might lead indirectly to new drugs. CHALLENGES TO BE MET Today we can use DNA technology to produce drugs that have im- portant clinical uses, and in the future we can expect products, as yet unimagined, that will flow from our increased understanding of the biological processes brought about by the use of recombinant-DNA technology as a research tool. However, further problems must be solved in order for there to be broad application of biotechnology to the pro- duction of proteins in the pharmaceutical industry. These challenging problems involve the following: Fermentation Regulation of protein production New host organisms, including mammalian cells Fermentation technology development Protein recovery and purification

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BIOTECHNOLOGY FOR HEALTH CARE 75 Many of the problems faced today in the application of recombinant DNA within the industry relate not to molecular biology and to how one genetically engineers a cell, but to how the production of protein in the fermentation vessel itself is regulated once that cell has been genetically engineered. We know today that there are proteins that probably will not be able to be produced economically in bacterial cells. We believe that there will be occasions in the future where mammalian cells will have to be used to produce those proteins. So the biotechnologists or bioengineers in the fermentation industry will have to learn to deal with mammalian cells on a large scale or in mass culture. It is my belief that the companies that develop their fermentation technology in general to the greatest degree will be the most successful in applying recombinant DNA and biotechnology. There will be need for great improvement in protein recovery and purification. Many of the techniques used today on production scale, for instance, to produce human insulin, really mimic the techniques of the laboratory. There is great opportunity for new innovation particu- larly in the area of protein recovery and purification. And finally, it appears that the organizations that will most success- fully apply biotechnology in the future will be the companies that can bring about the closest collaboration between the genetic engineer and the biochemical, electrical, and other engineering disciplines.

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