BTG 401 Immunology Lecture Notes PDF

# PMB 482: Basic Pharmaceutical Biotechnology and Immunology ## Lecture Note by Pharm. Oli Angus N (PhD) ### Course Outline: - Pharmaceutical immunology including but not limited to engineering antibodies for therapy - production of monoclonal antibodies, recombinant antibodies and antibody fragm...

# PMB 482: Basic Pharmaceutical Biotechnology and Immunology ## Lecture Note by Pharm. Oli Angus N (PhD) ### Course Outline: - Pharmaceutical immunology including but not limited to engineering antibodies for therapy - production of monoclonal antibodies, recombinant antibodies and antibody fragment. - Biotechnology in vaccines development - DNA vaccines, vaccine production by recombinant DNA for prevention of viral and bacterial infections; Clinical importance of recombinant proteins e.g. human insulin, growth hormones, interferon; Gene Therapy ## A. What is Immunity? In health science, immunity may be defined as 'the state of being protected from a disease especially an infectious disease'. Importantly, this particular state is invariably induced by having been exposed to the antigenic marker on a microorganism that critically invades the body or by having been duly immunized with a vaccine capable of stimulating the production of specific antibodies. ## Immunology - The study of the generation of an immune response - solely depends upon the prevailing interaction of three cardinal components of the immune mechanism, such as: immunogen stimulation, humoral immune system, and cellular immune system. - Since 1901 and as to date the epoch making discovery and spectacular evolution of immunobiotechnology' i.e., conglomeration of immune system variants, across the world hasrevolutionized not only the safer quality of life of human beings but also provided a broad spectrum ofnewer avenues in combating the complicated dreadful not-so-easy diseases of the present day. ## Immune Response In reality, the immune responses do refer to such processes whereby animals(including humans) give rise to certain specifically reactive proteins (known as 'antibodies') andadequate cells in response to a great number of foreign organic molecule and macromolecule variants. Based on the scientifically demonstrated proofs and evidences, the generalized immune responseessentially possesses four major primary characteristic features, such as: - **Discrimination:** It usually designates the 'ability of the immune system' to have a clear-cut discrimination between 'self' and 'non-self'; and, therefore, it invariably responds exclusively to such materials that happen to be foreign to the host. - **Specificity:** It refers to such a response that is extremely specific either solely for the inducing material or antigen to which the immune cells or antibodies would interact in a much prominent and greater strength. - **Anamnesis:** It most commonly refers to the critical ability to elicit a larger specific response much more rapidly on being induced by a second exposure' to the same very foreign antigen. It is also termed as the anamnestic response orthe immunologic memory - **Transferability by living cells:** Interestingly, the active immunity is observed to be exclusively transferable from one particular inbred animal specimen to another by the respective 'immune cells' or 'lymphocytes', and definitely not by immune serum which is capable of transferring temporarily the passive immunity, whereas the active immunity certainly needs the long-term regenerative ability of the living cells. ## B. What are the key concepts? - **Immunogens:** Are chemicals compounds that cause a specific immune response. - **Antigens:** Are chemicals compounds that bind to products of an immune response. When the antigens are recognized by antibody or activated cells, they can be eliminated by a specific immune response. - **Antibody:** Any of the complex glycoproteins (i.e. IgG, IgM, IgA, IgD) produced by B lymphocytes in response to the presence of an antigen. - **Adjuvants:** Are nonspecific substances, like alum, mineral oil, that essentially do possess the ability to prolong as well as intensify the ensuing immune response to a particular antigen on being injected simultaneously with the antigen. They profusely aid the immune response. - **Immunogen-antigens:** Substances associated with or secreted by parasitic microorganisms and are of molecular weight greater than 5Kilodaltons which can act as both immunogens and antigens. Molecular complexity is as important as molecular weight in determining the status of a compound as an immunogen. For a molecule to be immunogenic, it must contain protein or peptide. ## Types of Specific Immunity ### a. Acquired Immunity - Refers to the 'protection' an animal inherently develops against certain types of microorganisms or foreign substances. - It gets developed in the course of an individual's lifespan. - Acquired immunity may be summarized as shown below in Figure 2. | | | | | |------------------|--------------------------|------------------------------------|------------------------| | **Active** | **Naturally Acquired** | **Artificially Acquired** | **Passive** | | | Antigens enters the body | Antigens are Introduced in vaccines | Preformed antibodies in | | | naturally; body produces | body produces antibodies and | Immune serum introduced | | | antibodies and specializes | specialized lymphocytes | into body by injection | | | lymphocytes | | | ### b. Active Immunity - Refers to the specific immunity obtained from the development within the body of antibodies or sensitized T lymphocytes (T Cells) which critically neutralize or destroy the infective agent. - It may eventually result from the immune response to an invading organism or from inoculation with a vaccine essentially containing a foreign antigen. ### c. Cell-Mediated Immunity (or T-cell Mediated Immunity) - It has been duly observed that the regulatory and cytotoxic action of T cells during the specific immune response is known as the cell-mediated immunity. - However, the entire process essentially needs almost 36 hr to accomplish its full effect. - It is also called as T cell mediated immunity. - Interestingly, unlike B cells, T cells invariably fail to recognize the socalled foreign antigens on their own. - A foreign antigen is duly recognized by a macrophage whichengulfs it. - It displays part of the antigen on its surface next to a histocompatibility or 'self' antigen(macrophage processing). - Finally, the presence of these two markers together with the secretion of a cytokine, interleukin-1 (IL-1) by macrophages and other antigen-presenting cells duly activates CD4+/CD8 T cells (i.e., helper T cells), that categorically modulate the activities of other cells adequatelyinvolved in the immune response. - Thus, the CD4+T cells secrete interleukin-2 (IL-2), which stimulates the activity of natural killer cells (NK cells), cytotoxic T cells, and B cells; and ultimately promotes the proliferation of CD+T cellsin.order that the invading pathogen may be destroyed or neutralized effectively.. - Besides, GammaInterferon secreted by CD+T cells increases distinctly the macrophage cytotoxicity and antigenprocessing.. - However, the T-cell mediated immunity plays a significant and pivotal role in the rejectionof transplanted tissues and in 'tests for allergens'. i.e., the delayed hypersensitivity'reaction. ### d. Congenital Immunity - The congenital immunity refers to the immunity critically present at birth. - It may be either natural or acquired; the latter predominantly depends upon the antibodies solely received from the mother's blood. ### e. Herd Immunity - The herd immunity represents the immune protection duly accomplished via vaccination of a portion of a population that may eventually minimize the spread of a disease by restricting the number of potential hosts for the respective pathogen. ### f. Humoral Immunity (or B-cell Mediated Immunity) - Humoral immunity represents the immunity duly mediated by antibodies in body fluids e.g., plasma or lymph. - As these antibodies arè adequately synthesized and subsequently secreted by B cells that protect the body against the infection or the re-infection by common organisms, such as: streptococciand staphylococci, it is also known as B-cell mediated immunity. - In reality, the B cells are stimulatedby direct contact with a foreign antigen and differentiate into the plasma 'cells that·yield antibodies against the antigen; and the corresponding memory cells which enable the body to rapidly producethese antibodies if the same antigen appears'at a later time. - It is, however, pertinent to state here that B cell differentiation is also stimulated duly by interleukin-2(IL-2), secreted by the T4 cells, and by foreign antigens processed by macrophages. ### g. Local immunity - Is usually limited to a given area or tissue of the body. ### h. Natural immunity - Refers to the immunity programmed in the DNA, and is also known as the genetic immunity. - It has been observed that there are certain pathogens that fail to infect some species due to the fact that the cells are not exposed to appropriate environments, for instance: the 'measles virus' cannot reproduce in the canine cells ; and, therefore, dogs do have natural immunity to measles. ### i. Passive immunity - Specifically refers to the immunity acquired by the introduction of preformed, antibodies into an unprotected individual. - It may take place either through injection or in utero from antibodies that usually pass from the mother to the fetus via the placenta. - It can also be acquired by the newborn by ingesting the mother's milk. The two major segments of vertebrate immune system are: - Immunity associated with serum-transfer reflecting the activities of the humoral (antibody-mediated) immune system. - Immunity associated with transfer of lymphocytes reflecting the activities of the cell-mediated immune system. Nevertheless, these two aforesaid major segments exert their actions both individually, and together in order to safeguard the humans from ailment irrespective of their age, race, and gender. ## Immunological Memory The intensity of the humoral response is reflected by the 'antibody titer' (i.e. the total quantum of antibody present) in the serum. Soon after the very first initial contact with an antigen, the serum of the exposed person emphatically comprises of absolutely no detectable antibodies up to even several days at a stretch. However, one may distinctly notice a gradual rise in the 'antibody titer' (first and foremost. IgM antibodies are produced and subsequently IgG antibodies). Ultimately, a slow decline in antibody titer takes place. Importantly, the ensuing pattern. of decline duly designates the characteristic feature of a primary response to an antigen. However, the immune responses of the host get adequately intensified immediately after a second exposure to an antigen. Nevertheless, this secondary response is usually termed as memory or anamnestic response. It has been observed that there exists certain activated B lymphocytes that fail to turn into the so called antibody-producing plasma cells, but do persist and sustain as the long-lived memory cells. After a long span even stretching over to several decades, when such 'cells' are duly stimulated by the 'samme antigen', they invariably tend to differentiate rapidly into the much desired antibody-producing plasma cells. Actually, this ultimately affords the fundamental-basis of the secondary immune response.. ## Cells Of The Immune System ### 1. B lymphocytes (B cells) and T lymphocytes (T cells) - Are. the primary cells of specific immune response. - They are antigen' specific because they have specific antigen receptors embedded. in their. plasma membranes. - They have thousands of identical antibodies in their membranes that allow them to bind to a small group of chemically related antigens. - This group defines the antigen specificity of each B cells. - Different B cells have different antigen specificity. - B-cells that recognize specific antigens divide to form new B cells (memory B cells) and plasma cells: (antibody-forming cells), which secrete free, soluble" (humoral) antibody molecules into the extracellular fluids. - Virgin B cells have not responded to an antigen since their release into the circulation from the bone marrow. - Their membrane antibodies are of the immunoglobulin M (IgM) and D (IgD). - Memory B cells, are derived by cell division from another B cell. that has responded to an antigen. - Their membrane antibodies are of immunoglobulin classes A (IgA), E (IgE), G (IgG). ### 2. T lymphocytes (T cells) - Are the primary cells of specific immune response. - They are antigen specific because they have specific antigen receptors embedded in their plasma membranes. - They have two membrane proteins (a and ẞ or Y and 8), which define the antigen specificity of each T cell and several integral proteins known as Cluster of Differentiation 3 complex. - T cells are also called CD3+. - Each T cell has thousands of identical antigen receptors in its membrane. - Different "T cells of different antigens specificities differ in the conformation of their antigen receptors. - The antigens receptors of T cells do not recognize antigens, alone but also recognize peptide epitopes (fragments of antigen) that are chemically combined with Major Histocompatibility Complex (MHC) proteins on the surfaces of other body cells: - **MHC proteins are divided into two major classes, viz:** - Class I proteins - these are present on the surfaces of almost all body cells and - Class II proteins - these are present only on the surfaces of special Antigen-Presenting Cells (APCs). - T cells do not enter the circulation directly from bone marrow but first enter the thymus gland to mature. - Most developing T cells die in the thymus because they either do not recognize normal self-antigens or produce a response against normal self-antigens. - The T cells that are eventually released from the thymus into the circulation are virgin T ceils while the T cells that are that originate through cell division from the responses of other T cells are memory T cells or effector T cells. - Most T cells can be classified by the presence of a membrane glycoprotein known as CD4 - the helper, or TH cells or the presence of CD8 - the cytotoxic T lymphocyte (CTL), or TC cell. - TH cells can be divided into two functional groups: - TH1 and - TH2. - These have different functions in the immune response and regulate immune responses through the production interleukins - the proteins that act on cells in an autocrine, paracine or endocrine manner. - Tulcells activate other cells, including some TH cells,. To cells and macrophages. - They can also decrease antibody (Ab) production by inhibiting the formation of TH1cells, TH2 activate B cells to divide and produce Ab. - They can also inhibit the formation of TH1 cells. To cells are capable of killing cells that are infected. by viruses. - They do this through direct binding with the infected cells or through the release of cytotoxins. - Tr cells (also called T regulatory cells) have recently been described. - Most are CD4+ and some are CD8+. - All suppress immune response through the secretion of IL-10 and transforming growth factor ẞ (TGF-β): - CD4+ CD25+ Cells are also able to inhibit other T cells through direct contact.. ### 3. Natural killer (NK) cells - Are large, granularl lymphocytes without a specific T or B cells antigen receptors. - Their cytotoxicity is similar to that of CTL (Tc) cells. - They recognize and destroy tumor cells and are important for controlling viral infections prior to the development of adaptive immunity.. ### 4. Antigen-Presenting Cells (APCs) - Are essential for most immune responses and are found in the site at which these responses originate. - The best understood. APCs cells are the macrophages and dendritic cells of the lymph nodes, spleen and other lymphoid tissues. - Most immune responses within these organs begin with these cells present epitopes bound to their surface MHC class II molecules to TH cells and secrete cytokines as accessory signals. - APCs, especially macrophages, are able to control the type his of immune response generated. - They do by secreting different types of pathogens (e.g. extracellular and intracellular bacteria, and viruses). - Any cell in the body can act as an APC for immune response involving CTL cells. - Nucleated cells can present fragments of their surface MHC class I molecule to Tc (CD 8+) lymphocytes. - Both T and B cells continually circulate from the blood through the lymph nodes, spleen and other secondary lymphoid tissues and then back into the blood.. - If there is antigen present in the secondary lymphoid tissue that binds specifically to the receptor on the T or B cell, then an immune response can begin. ### 5. Neutrophils, Macrophages, eosinophils, basophils, platelets and mast cells - Assist in eliminating antigens from the body. - Their functions may be phagocytic, pro-inflammatory, cytotoxic, regulatory or a combination of these. ## Production of Monoclonal Antibodies for Therapeutic Use Monoclonal antibodies (mAb or moAb) are mono-specific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Mortoclonal antibodies have monovalent affinity, in that they bind to the same epitopes. Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology, and medicine. When used as medications, the non-proprietary drug name ends in -mab. Production of monoclonal antibodies involving human-mouse hybrid cells was described by Jerrold Schwaber in 1973. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies fuse them with healthy antibody-producing B-cells, and select the successfully fused cells. In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients. ### Hybridoma cell production (Hybridoma technology) Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a Rabbit Hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium in which only fused cells can grow is used. This is possible because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. By exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids requiring supplementation to survive. The selective culture médium is called HAT médium because it contains Hypoxanthine, Aminopterin, and Thymidine. This medium is selective for fused (Hybridoma) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (similar to a cancer cell). This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such 55 ELISA or Antigen Microarray, Assay) or immuno-dot blot. The most productive and stable clone is then selected for future use. - The hybridomas can be grown indefinitely in a suitable cell culture medium. - They can also be injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid. The medium must be enriched during in-vitro selection to further favour Hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement inedium such as briclone. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical. ### Purification of monoclonal antibodies After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and; as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture; and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other. The sample is first conditioned, or prepared for purification: Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultra-filtration or díalysis. Most of the charged impurities are usually anions such as nucleic acids and endotoxins. 1. These are often separated by ion exchange chromatography: - either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or - anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. - Various proteins can also. be separated out along with the anions based on their isoelectric point (pI). For example, albumin has a pl of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pl of 6.1. In other words, at, a given pH, the average charge of albumin molecules is likely to be more negative. Transferrins, on the other hand, have pl of 5.9, so cannot easily be separated out by this method. A difference in pl of at least 1 is necessary for a good separation. 2. Transferrins can instead be removed by size exclusion chromatography. - The advantage. of this purification method is that it is one of the more reliable chromatography techniques. - Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays telatively constant. - Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times. 3. A much quicker, single-step method of separation is **Protein A/G affinity chromatography**. - The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. - However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. - A low pH can break the bonds to remove the antibody from the colurnn. - In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. - Gentle elution. buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. - Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin.. 4. To achieve maximum purity in a single.step, **affinity purification** can be performed, using the antigen to provide exquisite specificity for the antibody. - In this method, the antigen used to generate the antibody is covalently attached to an agarose support. - If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective, attachment to a carrier protein, such as KLH dusing development and to the support for purification. - The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. - An elution with a low pH buffer or a more gentle high salt elution buffer is then used to recover purified antibody from the support. 5. To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. - Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. - The appropriate level of salt is added in order to achieve the best separation. - Excess salt must then be removed by a desalting method such as dialysis. The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates fhe amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present. ### Antibody heterogeneity Product heterogeneity is common to monoclonal antibody and other recombinant biological production and is typically introduced either upstream during expression or downstream during manufacturing. These variants are typically aggregates, deamidation products, glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions. These seemingly minute changes in monoclonal antibody's structure can have a profound effect on preclinical stability and process optimization as well as therapeutic product potency, bioavailability, and immunogenicity. The generally accepted method. of purification of monoclonal antibodies includes capture of the product target with Protein A, elution, acidification to inactivate potential Mammalian viruses, followed by cation exchange chromatography, and finally anion exchange chromatography. Displacement chromatography, has been used to identify and characterize these often unseen variants in quantities that are suitable for subsequent.preclinical evaluation regimens such as animal pharmacokinetic studies. Knowledge gained during the preclinical development phase is critical for enhanced understanding of product quality and provides a basis for risk management and increased regulatory flexibility. The recent Food and Drug Administration's Quality by Design initiative attempts to provide guidance on development and to facilitate design of products and processes that maximizes efficacy and safety profile while enhancing product manufacturability. ## Recombinant Monoclonal Antibodies Production The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. The phage antibody libraries are a variant of the phage antigen libraries first invented by George Pieczenik. These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale: ## Chimeric Antibodies Definition: Antibodies that is composed of genetically different tissues, either naturally or as a result of a laboratory procedure. Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse; not human antibodies. While structurally similar, differences between the two were sufficient to invoke an immune response when murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects, and the production. of human anti-mouse antibodies (HAMA). In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody: was merged with human antibody-producing DNA in living cells. The expression of this chimeric DNA through cell culture yielded partially mouse, partially human monoclonal antibodies. For this product, the descriptive terms "chimeric" and "humanised" monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process. ## 'Fully' Human Monoclonal Antibodies Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of 'fully' human antibodies to avoid some of the side effects of: humanized or chimeric antibodies. Two successful approaches have been identified: transgenic mice and phage display. Transgenic mice technology is by far the most successful approach to making 'fully' human monoclonal antibody therapeutics. Seven (7) of the 9 'fully' human monoclonal antibody therapeutics on the market were derived in this manner. Transgenic mice have been exploited by a number of commercial organisations: - Medarex - who marketed their UltiMab platform. Medarex were acquired in July 2009 by Bristol Myers Squibb. - Abgenix - who marketed their Xenomouse technology. Abgenix were acquired in April 2006 by Amgen. - Regeneron's VelocImmune technology. - Kymab - who market their Kymouse technology. One of the most successful commercial organisations using phage display technology was Cambridge Antibody Technology (CAT). Scientists at CAT demonstrated that phage display could be used such that variable antibody domains could be expressed on filamentous phage antibodies. ## Applications Monoclonal antibodies have been generated and approved to treat cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection. Other applications of monoclonal antibodies include: ### 1. Diagnostic tests Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test and immuno dot blot tests detect the protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells. ### 2. Monoclonal antibody therapy - **Cancer treatment:** One Cossible treatment for cancer involves monoclonal antibodies that target only cancer cell specific antigens and induce an immunological response against the target cancer cell. - Such mÄb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate. - It is also possible to design bispecific monoclonal antibodies that can bind with their Fab regions to both target antigen (in this case, cancer cells) and to a conjugate or effector cell. - Every intact antibody can bind to cell receptors or other proteins with its Fc region. Monoclonal antibodies for cancer, include: - ADEPT, antibody directed enzyme prodrug therapy; - ADCC, antibody dependent cell-mediated cytotoxicity; - CDC, complentent dependent cytotoxicity; - MAb, monoclonal antibody;. - scFv, single-chain Fv fragment. - **Autoimmune diseases:** Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind to and inhibit TNF-a. - Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help prevent acute rejection of kidney transplants. - Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma. - **Pathogen neutralization and antiviral therapy.** Antibody binding can directly and effectively block the activity of many pathogens, often without requiring Fc-mediated cytotoxicity. - Indeed, this has always been the promise of antibody-mediated viral neutralization. - The firsi monoclonal antibody for the treatment of viral disease, Synagis, 'was approved by the FDA in 1998 (Table 1). - Synagis is a humanized antibody used for the prevention of severe respiratory syncytial virus (RSV) disease. - Despite this success, and the wide range of antibodies available against human immunodeficiency type 1 (HIV) and herpes simplex virus (HSV), the use of recombinant antibodies as therapeutics for viral infection has been limited. - **Intracellular antibodies**. Antibody fragments can be expressed as intracellular proteins, typically as scFvs termed intrabodies, and equipped with targeting signals either to neutralize intracellular gene products or to target cellular pathways. - Intrabodies also have important antiviral potential, particularly through their targeting of intracellular action to mandatory viral proteins such as the Vif, Tat or Rev Components of HIV72. - Antibody frameworks have been adapted that substantially improve expression levels and solubility in the intracellular reducing environment.. - The expression of intrabodies in vivo can be encoded into gene therapy vectors, and this could ultimately be theit most powerful clinical application. - **T-cell applications:** Troy-bodies are engineered vaccine antibodies containing typ T-cell epitopes to enhance antigen presentation. - Troy-bodies effectively target antigen presenting cells (APCs) and, after processing, expose cryptus T-cell epitopes to direct T-cell activation. - In the preferred format, the Fv domain provides APC specificity and the C domains encode the cryptic T-cell epitopes. - These new vaccines can be redesigned to target many different APCs and enhance immunity to many different T-cell epitopes. - Alternativè vaccine strategies include the use of engineered, APC-targeted antibodies that direct adenoviruses to deliver vaccine-inducing epitopes as a gene therapy capsule and B7-targeted scaffolds (scFv and VL domains) that enable antigen-loading of dendritic cell.. ## Vaccine Vaccines belong to the category of immunobiologicals - products that are produced by or derived from a living organism: Immunobiologicals include a 'variety of products, such as vaccines, immunoglobulins, monoclonal antibodies, and antisera. The characteristic feature of vaccines is that these preparations are capable of inducing a specific and active immunity against an infecting agent or its toxin. A vaccine is an immunological product that produces immunity from a disease and can be administered through needle injections, by mouth, or by aerosol. A vaccination is the administration of vaccine for the purpose of producing immunity in the body against infection. Immunization is the process by which a person or animal becomes protected from a disease. Vaccines cause immunization, and there are also some diseases that cause immunization after an individual recovers from the disease. ## Biotechnology in Vaccines Development- Ina Vaccines, Vaccine Production by Recombinant DNA for Prevention of Viral and Bacterial Infections ### Vaccines through Biotechnology Biotechnology is used in four different ways in the development of vaccine: - Use of cloned genes for the synthesis of antigens - Use of knock-out genes: - Use of Synthetic peptides as vaccines - Use of anti-idiotype antibodies ### Use of cloned genes for the synthesis of antigens: Hundreds of genes in eukaryotes have been cloned from genomic DNA or from cDNA. Through genetic engineering (recombinant DNA technology), scientists can isolate specific genes and insert them into DNA of certain microbes or mammalian cells; the microbes or cells become living factories, mass producing the desired antigen. Then, using another, product of biotechnology, a monoclonal antibody that recognizes the antigen, it is separated from all the other material produced by le microbe or cell. This technique has been used to produce immunogenic but safe segments of the hepatitis B virus and the malaria parasite. The HBV genome was cloned in the plasmid pBR322 followed by its propagation in. E. coli. The antigens produced from this clone reacted with hepatitis B core antibody (HBAb) which has. been used to produce hepatitis B vaccine. Efforts are on to use this method to produce an Anti-malarial vaccine. Malarial parasite Plasmodium falciparum is the malarial parasite which has become a major threat to human health which spreads by the mosquito bite. In human body, the malarial parasite passes through several antigenically distinct phases, viz. sporozoite, merozoite, gametocytes etc. The vaccines can be developed to control any of these phases hence we can have antisporozoite vaccine, antimerozoite vaccine etc. Out of all these, considerable progress has been made in the making of antisporozoite vaccine due to cloning of gene meant for circumsporozoite (CS) protein. This protein was, obtained directly from DNA of erythrocytic form of parasite, rather than as cDNA from mRNA. This cloned gene may, in course of time, lead to the synthesis of vaccine by synthesizing CS protein by clone gene. In another approach, scientists have inserted genes for desired antigens into the DNA of the vaccinia virus, the large cowpox virus familiar for its role in smallpox immunization. When the reengineered vaccinia virus is inoculated, it stimulates an immune reaction to both the vaccinia and the products of its passenger genes. These have included, in animal experiments, genes from the viruses that cause hepatitis B, influenza, rabies, and AIDS. ### Use of knock-out genes: Instead of adding a gene, some scientists have snipped a key gene out of an infectious organism. Thus crippled, the microbe can produce immunity but not disease. This technique has been tried with a bacterium that causes the severe diarrheal disease cholera; such a vaccine is commercially available against a virus disease of pigs. ### Use of Synthetic peptides as vaccines Vaccines can also be prepared through short synthetic peptide chains. There are several ways by which these can be used as vaccines: - A totally different approach to vaccine development lies in chemical synthesis. Once scientists have isolated the gene that encodes an antigen, they are able to determine the precise sequence of amino acids that make up the antigen. They then pinpointed small key areas on the large protein molecule that is responsible for the immunogenic response noting their three dimensional structure, and assembled it chemical by chemical. It is this three dimensional structure (and notnecessarily the amino acid sequence) of à protein that is responsible for the immunogenic response. E.g. in Foot and Mouth Disease virus (FMDV), the amino acid 114-160 of virus polypeptide can produce antibodies which can neutralize FMDV and provide protection. The region of 201-213 amino acids of the same protein also could neutralize FMDV hence it has been shown that small synthetic peptides representing these regions of proteins can show immunogenic response and can be used for the development of vaccine.. - The immunogenic region of protein can also be located by gene coding for the protein. E.g. in Feline leukaemia virus, the clone gene of an immuriogenic protein was cut into fragments by DNAase I and then cloned in lambda phage. Phage colonies (plaques) with different cloned fragments are screened with a specific monoclonal antibody that neutralizes the pathogen. The fragments which react with antibody must. be synthesizing the immunogenic peptide fragments which can be sequenced. Using this method it was possible to identify a 14 amino. acid immunogen of the envelope protein of Feline leukaemia virus (FL