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Microbial Production of Therapeutic Agents: Before the development of recombinant technology, most human protein pharmaceuticals were available in: Limited quantities They were costly to produce and In a number of cases, their biological mode of action was not well characterized After the development of recombinant DNA technology, it was heralded/indicated as a means of producing a whole range of possible human therapeutic agents in sufficient quantities for both efficacy testing and eventual human use This forecast has turned out to be true Today, the genes mostly cDNAs for over 1,000 different proteins that are potential human therapeutic agents have been cloned Most of these sequences have been expressed in host cells and currently around 500 are undergoing clinical trials for the treatment of various human diseases More than 250 of these “biotechnological drugs” have been approved for use in the United States or the European Union Table 10.1 Glick 4th Ed However, it will be several years before many of the other proteins are commercially available, because medical products must first be tested rigorously in animals and then undergo through human trials before being approved for general use It has been estimated that in 2006 the annual global market for human recombinant protein drugs was about $ 60 billion and ten “block buster” drugs constitute nearly half of these sales In 2006, rituzimab (Rituzan), a monoclonal antibody used to treat individuals with non-Hodgkin lymphoma, generated nearly $ 4 billion in sales While various forms of recombinant insulin generated around $ 2.5 billion The development of treatments preventive procedures and cure for human diseases was the outstanding contribution of medicine and science to human well being in 20th century This process, however, is a continuous one So called old diseases like tuberculosis can reappear if preventive measures are relaxed or if resistant organism arise The idea of using antibodies as therapeutic agents has come to fruition in past several years and specific antibodies are being tested to attack: toxins bacteria viruses or even cancer cells An antibody may be viewed as a target-seeking missile or as a magic bullet that either can directly neutralize an offending agent or if equipped with a warhead or poison arrow can destroy a specific target cell Unfortunately, despite their theoretical promise, the use of antibodies for preventing or treating diseases and other conditions has been limited However, recently, the development of recombinant DNA technology and means of producing monoclonal antibodies has combined with rapidly growing understanding of the molecular structure and functions of immuno-globulin molecules to reactivate interest in using specific antibodies to treat various diseases Pharmaceuticals: Engineering Human Interferon (IFN); Interferons can be classified into three different groups on the basis of chemical and biological properties • IFNα and IFNβ: i. Synthesized in cells that have been exposed to viruses/viral DNA ii. • IFNα encoded by a family of 15 non-allelic genes while IFNβ by a single gene IFN-γ: Synthesized in response to cell growth stimulating agents and encoded by a single gene IFNα, β and γ have subtypes with different specificities For Example: • Anti viral activities of IFNα2 and α1 approximately same when assess with a virus challenged bovine cell line but • IFNα2 is seven times more effective than IFNα1 when human cells are treated with virus • IFNα2 30 times less effective than IFNα1 when mouse cells are used in this assay Table 10.3 Glick 2nd Ed Attempts have been made to engineer IFNs with combined properties In one study, hybrid genes from IFNα2 and α3 were constructed in an effort to create proteins with novel IFN activities Fig. 10.2 Glick 3rd Ed Hybrids expressed in E. coli Resultant proteins were purified and examined for various biological functions Greater activity then parental molecules noted when tested for the extent of protection of mammalian cells in culture Many of hybrid IFNs induced test cells to synthesize 2’-5’ oligoisoadenylate synthetase This enzyme generates 2’- 5’ oligoisoadenylate which, in turn, activates a latent cellular endoribonuclease cleave viral mRNA Other hybrids had an anticancer activity against human cancers This activity was greater then that of the parental molecules The creation of these hybrid interferon demonstrates that new potential therapeutic molecules can be constructed by combining functional domains from related gene Engineering Human Growth Hormones; The strategy of designing proteins by either functional domain shuffling or site-directed mutagenesis can be used to augment or constrain the mode of action of a protein Native human growth hormones (hGH) bind to both growth hormones and prolactin receptors on a number of different cell types To avoid unwanted side effects during therapy it is desirable that hGH bind only to GH receptors Fig. 10.3 Glick 3rd Ed Enzymes: DNase 1; Cystic fibrosis most common fatal hereditary disease among Caucasians Approximately 30,000 diagnosed cases in USA and 23,000 in Canada and Europe It is estimated that a mutant cystic fibrosis gene is carried by 1 in 29 Europeans, 1 in 65 African Americans and 1 in 150 Asians Individual with this disease are highly susceptible to bacterial infection in their lungs Antibiotic treatment of patients who have these recurring infections eventually leads to the selection of the antibiotic resistant bacteria Presence of bacteria, some alive and some lysed causes the accumulation of a thick mucus in the lungs of these patients, making breathing very difficult A portion of the thick mucus in the lungs is the result of the DNA that is released when bacterial cells are lysed To address this problem, Scientists at the USA Biotechnology Company, Genentech isolated and expressed the gene for the enzyme DNAse 1 which can hydrolyzed long polymeric DNA chain into much shorter oligonucleotides Then they delivered the purified enzyme in an aerosol to the lungs of the patients with cystic fibrosis What DNAse treatment has done? The DNAse treatment acted to decrease the viscosity of the mucus in the lungs and made it easier for these patients to breathe While this treatment is not cure for cystic fibrosis, it nevertheless relieves the most severe symptoms of the disease in most of patients This enzyme has been approved in 1994 by USA Food and Drug Administration Sale is predicted approximately $ 100 million per annum by around the year 2000 Fig 17.3 Brum Fig 17.7 Glick 3rd Ed Monoclonal Antibodies as Therapeutic Agents: Today with the advent of hybridoma methodology, antibodies are once again seen as potential therapeutic agents In fact, a number of monoclonal antibodies have been approved for treating human diseases Table 10.3 Glick 4th Ed This technique can be used to maintain a continuous supply of pure mono-specific antibody Fig 10.13 Glick 3rd Ed complementaritydetermining regions Papian - Two Fab and one Fc Preventing Rejection of Transplanted Organisms: In the 1970s, passive immunization was considered as a way of preventing immunological rejection of a transplanted organ The rationale was to administer a specific antibody to the patients that would bind to certain lymphocytes and diminish the immune response directed against the transplanted organ In this effort, OKT3, the mouse monoclonal antibody was first approved in 1986 by the USA Food and Drug Administration For use as an immunosuppressive agent after organ transplantation in humans Mechanism: Lymphocytes that differentiate in the thymus are called T cells Various members of the T-cells population act as immunological helper and effector cells are responsible for organ rejection OKT3 binds to cell surface receptor CD3 which is present on all T cells This action prevents a full immunological response and spares transplant organ from rejection Immuno-supression by this means was reasonably effective but there were some side effects including fever and rash formation as anticipated. Why….? Chemically Linked Monoclonal Antibodies: Drugs that are very effective when tested in vitro – usually in culture- are often much less potent in vivo This apparent loss of potency is due to the drug’s not reaching its targeted site at a concentration sufficient to be effective Increasing the dose of a drug is not the answer to this problem, because high drug concentrations often have deleterious side effects Moreover, to avoid unwanted side effects, many drugs administered at lower than optimal levels, thereby decreasing their efficacy A number of different strategies may be used to enhance the delivery of a drug to its target site: Drug may be encapsulated in liposomes i.e. particles in which drug is surrounded by a specific lipid surface, that can be targeted to certain organs Fig 2.17 Lodish 3rd Ed Fig 10.14a Glick 3rd Fig 10.14b Glick 3rd The majority of natural death in North America and Europe are the result of blockage of a cerebral/coronary artery by the blood clot i.e. thrombus A thrombus consists of a network of fibrin, a blood clotting agent, which is formed in response to defect in the walls of a blood vessel Under natural conditions, plasmin degrades the fibrin in a blood clot and dissolves the clot Plasmin is a serine protease and is produced by the activation of plasminogen by plasminogen activator In many instances, however, arterial blockage occurs when this biological system does not remove blood clots efficiently Thus, it was reasoned that plasminogen activators could be used as therapeutic agents to induce higher levels of plasmin, which in turn, would efficiently remove the arterial thrombi, thereby reducing their impact on brain and heart arteries Fig 10.15 Glick 3rd Ed Fig 10.16 Glick 3rd Ed Human Monoclonal Antibodies: Although the initial studies of immunotherapeutic agents were promising, there are drawbacks to chemical coupling and the use of a nonhuman monoclonal antibody Not only are the yields of chemically linked molecules low but the coupling occurred at random sites The chemical coupling procedure may also inactivate the enzyme activity of the plasminogen activator or other protein that may be used Finally as noted previously, if the therapy requires multiple treatments, the antibody component should be from human source to prevent immunological cross-reactivity and sensitization of the patient It is very difficult to create specific non-cross reactive antibodies because of problems associated with obtaining human monoclonal antibodies by conventional hybridoma techniques Therefore, other approaches for obtaining human monoclonal antibodies were devised: It may possible to introduce cells of the human immune system into mutant mouse strain that lacks for most parts, its own natural immunological cell repertoire Transplantation of human immune system stem cells, such a mouse, with severe combined immunodeficiency (SCID mouse) As a result mouse acquires a human cell immune system and In response to challenge by antigen, can produce human antibodies It may also to introduce human immunoglobin genes into the germ line of mice to create transgenic mice that would produce a human immunoglobin after immunization with particular antigen Both methods are laborious. Therefore, genetic engineering is now used to create: to create both human therapeutic antibodies and effective dual-function proteins that have the ability to bind a target and then destroy it Hybrid Human – Mouse Monoclonal Antibodies: The modular nature of antibody functions has made it possible to convert a mouse monoclonal antibody into one that has some human segments but still retains its original antigenbinding specificity This hybrid molecule is called a chimeric antibody or humanized antibody depending which portion of the mouse antibody are remove Fig 10.17 Glick 3rd Ed Fig 10.18 Glick 3rd Ed Fig 10.19 Glick 3rd Ed To date, with this procedure, more than 50 different monoclonal antibodies have been humanized While this technology is clearly effective and widely applicable but time consuming and expensive Single-Chain Antibody; In addition to producing Fv fragment, attempts have been made to determine whether a single protein chain consisting of only VL and VH domains would form a functional antigen binding molecule? Computer simulations of 3-dimensional structure of a potential single chain antibody showed that VL and VH domains have to be separated by a linker peptide to assume the correct conformation for antigen binding Example: - Humanized antibody directed against the surface of human colon cancer cells was tested in patients with colorectal cancer - Remained in blood system about 6X longer - Expended the period of effectiveness - Only one patient/10 showed mild response - Starting with a rodent Hybridoma cell line, cDNA for the L and H can be isolated. - The variable regions of the cDNAs can be amplified by PCR On the basis of these design constraints, DNA constructs VL and VH sequences from a cDNA of a cloned monoclonal antibody were each ligated to a chemically synthesized DNA linker fragment in the order VL-linker-VH After expression in E. coli, the single chain protein was purified and both its affinity and specificity were found to be equivalent to those of the original intact monoclonal antibody Single chain antibody may be used for a variety of therapeutic and diagnostic applications in which Fc effector's functions are not required and When a small size is an advantage as speeds antibody uptake and facilitates a more even distribution in solid tumors Fig 10.25a Glick 3rd Ed Fig 10.25b Glick 3rd Ed x----------------------- x ------------------------- x------------------------- x