MONOCLONAL ANTIBODIES ( MAB )

1. MONOCLONAL ANTIBODIES BY NADIKATLAANUSHA M.Pharm

2. CONTENTS INTRODUCTION CLASSIFICATION OF ANTIBODIES TYPES OF MONOCLONAL ANTIBODIES (mAb) • Murine Monoclonal Antibodies • Chimeric Monoclonal Antibodies • Humanized Monoclonal Antibodies • Human Monoclonal Antibodies STEPS INVOLVED IN PRODUCTION OF mAb BY HYBRIDOMA TECHNOLOGY PROPAGATION OF MAB BY: In vitro methods: a) Batch tissue culture method b) Semi permeable membrane method In vivo methods: Mouse ascites method EVALUATION OF MAB a) Transchelation Challenge Test b) Immunoreactivity studies c) Micro ELISA system d) Animal biodistribution study APPLICATIONS OF MAB a) Diagnostic Application b) Catalytic mAb (Abzymes) c) Therapeutic Application PROBLEMS ASSOCIATED WITH mAb’s CONCLUSION ANUSHA NADIKATLA

3. INTRODUCTION A membrane will have more than one single antigen to which T helper cell surface proteins can bind. Alternatively, some antigens may have more than one binding sites (‘epitopes’) ANUSHA NADIKATLA

4. All of the bound T-helper cells become ‘ACTIVATED’ Therefore, several different antibodies will be produced against each pathogen (i.e. Polyclonal) To target an antigen accurately, we need “One specific antibody” i.e. “MONOCLONAL” antibody. ANUSHA NADIKATLA

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7. ANTIBODY An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses. Antibody contains 2 light chains and two heavy chains joined together by disulfide bonds. Each heavy chain contains carbo- hydrate residue. Bottom trunk portion is known as constant region (Fc). The upper arms are the antigen binding regions (Fab) known as variable region. This region in turn has several “hypervariable” regions known as complementarity determining regions(CDR) which show greater variability than the rest of the variable region. ANUSHA NADIKATLA

8. The antibody recognizes a unique molecule of the pathogen, called an antigen, via the Fab's variable region. Each tip of the "Y" of an antibody contains a paratope(analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision. ANUSHA NADIKATLA

9. FUNCTIONS OF ANTIBODY Antibodies have two major functions: Recognize and bind antigen Induce immune responses after binding VARIABLE REGION CONSTANT REGION The variable region mediates binding. Affinity for a given antigen is determined by the variable region. The variable region confers absolute specificity for an antigen. The constant region mediates immune response after binding. Different classes of constant regions generate different isotypes. Different isotypes of antibody have differing properties ANUSHA NADIKATLA

10. CLASSIFICATION OF ANTIBODIES POLYCLONALANTIBODIES MONOCLONALANTIBODIES ANTIBODY FRAGMENTS CHIMERIC ANTIBODIES HUMANIZED ANTIBODIES BISPECIFIC ANTIBODIES ANUSHA NADIKATLA

11. POLYCLONALANTIBODIES If an animal is immunized with a protein, a wide array of B cells will be stimulated to produce anti-protein antibodies. They are a combination of immunoglobulin molecules secreted against a specific antigen. Polyclonal antibodies are a mixture of antibodies with different antigen binding sites that may bind to different epitopes or antigens of the immunizing agent with varying affinities. Each identify a different epitope. They may be of different antibody classes. ANUSHA NADIKATLA

12. An antibody binds to a specific region on an antigen called an epitope. A single antigen can have multiple epitopes for different, specific antibodies. ANUSHA NADIKATLA

13. A mixture of antibodies - all bind to epitopes of the original antigen. Some bind with higher affinity than others. ANUSHA NADIKATLA

14. MONOCLONALANTIBODIES (mAb) Monoclonal antibodies are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study. To produce the desired mAb, the cells must be grown in either of two ways: by injection into the abdominal cavity of a suitably prepared mouse or by tissue culturing cells in plastic flasks. ANUSHA NADIKATLA

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16. CHARACTERISTICS OF POLYCLONALAND MONOCLONALANTIBODIES MONOCLONALANTIBODIES POLYCLONALANTIBODIES Derived from a single B cell clone Derived from different B lymphocytes cell lines mAb offer reproducible, predictable & potentially inexhaustible supply of Ab with exquisite specificity Batch to batch variation affecting Ab reactivity & titre Expensive production Inexpensive production Long production time Rapid production Large quantities of specific antibodies Large quantities of nonspecific antibodies Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen Production is continuous and uniform once the hybridoma is made Different batches vary in composition Enable the development of secure immunoassay systems Not powerful tools for clinical diagnostic tests ANUSHA NADIKATLA

17. ANTIBODY FRAGMENTS: Earliest MoAb’s were of nonhuman origin which are immunogenic and exhibit human antimouse response(HAMA).To overcome this problem, antibody was cleaved into Fc and Fab fragments by papain digestion. The Fab fragments are less immunogenic than intact antibodies, penetrate more into tumour cells, longer halflife. But, Antigen binding capacity is lost up to some extent. CHIMERIC ANTIBODIES: It contains Fc region of human immunoglobulin (IgG) and Fab regions are murine (mouse) origin. These are slightly immunogenic. HUMANIZED ANTIBODIES: These are produced by rDNA technology. Majority of antibody framework is human in origin, but the CDR’s (responsible for antigen binding) are murine. BISPECIFIC ANTIBODIES: Each of the two arms, are specific for two different antigens i.e. Target 1, Target 2 ANUSHA NADIKATLA

18. MONOCLONAL ANTIBODIES (mAb) ANUSHA NADIKATLA

19. MONOCLONALANTIBODIES (mAb) Monoclonal antibodies are specific antibodies produced by fusing B-cells (B-lymphocytes) derived from a single ancestral B-cell with a tumour (myeloma) cell. These cultures of B-cells are called Monoclonal as they are derived from a single ancestral B-cell. These cells are used to harvest single kind of antibodies called as Monoclonal antibodies. They are highly specific and offer more consistent efficacy. Monoclonal antibodies (mab) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. ANUSHA NADIKATLA

20. TYPES OF MONOCLONAL ANTIBODIES (mAb) 1. Murine Monoclonal Antibodies 2. Chimeric Monoclonal Antibodies 3. Humanized Monoclonal Antibodies 4. Human Monoclonal Antibodies ANUSHA NADIKATLA

21. MURINE MONOCLONAL ANTIBODIES A murine antibody is one of which both chain types are of mouse origin. A murine antibody is identified by the pre-stem -o- in its INN. Major problems associated with murine antibodies include Reduced stimulation of cytotoxicity Formation of complexes after repeated administration Allergic reactions Anaphylactic shock ANUSHA NADIKATLA

22. CHIMERIC MONOCLONAL ANTIBODIES Chimeric Ab are obtained by genetically fusing the mouse variable domains to human constant domains [Boulianne et al.,1984; Morrison et al., 1984;Wright et al., 1992]. A chimeric antibody is one of which both chain types are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (V-D-J-REGION) (originating from one species other than human, or synthetic) linked to a constant region (C- REGION) of human origin. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource). ANUSHA NADIKATLA

24. mAb are genetically engineered using a molecular approach. Variable regions are Isolated using polymerase chain reaction (PCR). A chimeric antibody is identified by the pre- stem -xi- in its INN. Issues with Chimeric mAb: There are problems that can arise from using mouse-human Ab. sometimes the body may elicit an anti-chimeric Ab in the present of these genetically engineered Ab. ANUSHA NADIKATLA

26. HUMANIZED MONOCLONAL ANTIBODIES A humanized antibody is one of which both chain types are humanized as a result of antibody engineering. A humanized chain is a chain in which the complementarity determining regions (CDR) which are the responsible for antigen binding within the variable regions, are foreign (originating from one species other than human, or synthetic) whereas the remaining chain is of human origin, creating ‘‘CDR-grafted’’ or ‘‘humanized’’ antibodies. This is, in essence a human Ab with small segments containing mouse Ab genes. By extension an antibody is described as humanized if more recent protocoles were used for the humanization. A humanized antibody is identified by the pre-stem -zu- in its INN. ANUSHA NADIKATLA

27. HUMAN MONOCLONAL ANTIBODIES A human antibody is one of which both chain types, and the J chain in the case of polymeric antibodies, are of human origin. A human antibody is identified by the pre-stem -u- in its INN. Note that, in the case of polymeric antibodies, the INN name is only based on the immunoglobulin chain origin. The monoclonal antibodies produced by using mice are quite suitable for in vitro use. However, their administration to humans is associated with immunological complications, since they are foreign to human body. Production of human monoclonal antibodies is preferred. However, it is difficult to produce human MAbs by conventional hybridoma technology. Human antibodies directly from humans! It has proven technically much more difficult to immortalize and clone human B-cells and human hybridomas. Also it raises ethical problems of immunizing human donors ANUSHA NADIKATLA

28. POTENTIAL ADVANTAGES OF MONOCLONAL ANTIBODIES Single matched genetic source of antibody. Prevention of allogeneic immune response to mixed sequences in non-antigen-binding regions. Producing clone can be amplified to produce unlimited quantities of monoclonal as a pharmaceutical source. Antibody coding information can be engineered to precise sequential specifications to produce desired target binding to avoid adverse immunological reactions. Monoclonal antibodies truly represent a homogeneous state of a single molecular species. Each MAb is specific to a given antigenic determinant. This is in contrast to the conventional antiserum that contains polyclonal antibodies. The wide range of applications of MAbs is described later. ANUSHA NADIKATLA

29. Measuring protein and drug levels in serum. Typing tissue and blood. Identifying infectious agents. Identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas. Identifying tumor metastasis. Identifying and quantifying hormones. Immunoaffinity Purification ANUSHA NADIKATLA

30. LIMITATIONS OF MONOCLONAL ANTIBODIES Hybridoma technology is laborious and time consuming. MAbs are produced against a single antigenic determinant; therefore, they cannot differentiate the molecule as a whole. Sometimes, they may be incapable of distinguishing groups of different molecules also. The presence of retroviruses as a part of the mammalian chromosomes is a common occurrence. Mice used in MAb production carry several viruses (adenovirus, hepatic virus, retrovirus, reovirus, cytomegalovirus, thymic virus). The presence of some of these viruses has been detected in the hybridomas. This poses a great danger, since there is no guarantee that MAb produced is totally virus-free, despite the purification. For this reason, US Food and Drug Administration insists that MAb for human use should be totally free from all pathogenic organisms, including viruses. ANUSHA NADIKATLA

31. STEPS IN THE PRODUCTION OF MONOCLONAL ANTIBODIES 1) PRODUCTION OF MAB BY HYBRIDOMA TECHNOLOGY: The generation of mAB producing cells requires the use of animals, usually mice. The procedure yields a cell line capable of producing one type of antibody protein for a long period. A tumor from this “immortal” cell line is called a HYBRIDOMA. 2) PROPAGATION OF MAB BY: In vitro methods: a) Batch tissue culture method b) Semi permeable membrane method In vivo methods: Mouse ascites method ANUSHA NADIKATLA

32. SEQUENCE FOR MONOCLONALANTIBODY PRODUCTION BY HYBRIDOMA TECHNOLOGY 1) Immunize animal (mouse or rabbit) 2) Screening of mice for antibody production and Isolate spleen cells (containing antibody-producing B cells) 3) Preparation of myeloma cells 4) Fusion of myeloma cells with immune spleen cells (e.g. using PEG - polyethylene glycol) 5) Allow unfused B cells to die. Add HAT culture to kill unfused myeloma cells. 6) Cloning of hybridoma cell lines by “Limiting dilution” or Expansion & stabilisation of clones by ascites production. 7) Screen supernatant of each clone for presence of the desired antibody (ELISA). 8) Grow the chosen clone of cells in tissue culture indefinitely. 9) Harvest antibody from the culture supernatant. 10)Characterization and Storage. ANUSHA NADIKATLA

34. STEP 1: IMMUNIZATION OF MICE AND SELECTION OF MOUSE DONORS FOR GENERATION OF HYBRIDOMA CELLS The very first step in hybridoma technology is to immunize mice with appropriate antigen that is prepared for injection either by emulsifying the antigen with Freund’s adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. The antigen can be whole cells, membrane fragment, or complex molecules. The injections at multiple sites are repeated several times. This enables increased stimulation of B-lymphocytes which are responding to the antigen. ANUSHA NADIKATLA

35. Three days prior to killing of the animal, a final dose of antigen is intravenously administered. The immune-stimulated cells for synthesis of antibodies have grown maximally by this approach. The concentration of the desired antibodies is assayed in the serum of the animal at frequent intervals during the course of immunization. Mice serum’s are screened using various techniques such as ELISA. When the serum concentration of the antibodies is optimal, the animal is sacrificed. The spleen is aseptically removed and disrupted by mechanical or enzymatic methods to release the cells. The lymphocytes of the spleen are separated from the rest of the cells by density gradient centrifugation. ANUSHA NADIKATLA

36. STEP 2: SCREENING OF MICE FOR ANTIBODY PRODUCTION After immunization, blood samples are obtained from mice for measurement of serum antibodies. Serum antibody titer is determined with various techniques, such as ELISA, flow cytometry. If the antibody titre is high – cell fusion can be performed. If the titre is too low – mice can be boosted until an adequate response is achieved. If the titre is high enough – mice are commonly boosted by injecting antigen without adjuvant. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production immunization of mice. ANUSHA NADIKATLA

37. STEP 3: PREPARATION OF MYELOMA CELLS Myeloma cells are immortalised cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine aminopterin thymidine(HAT) selection medium used after cell fusion. A week before cell fusion,myeloma cells are grown in 8- azaguanine. ANUSHA NADIKATLA

38. STEP-4: FUSION OF MYELOMA CELLS WITH IMMUNE SPLEEN CELLS Single spleen cells from the immunized mouse are fused with the previously prepared HGPRT defective myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells & myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse for a short period (a few minutes), since it is toxic. PEG is removed by washing and the cells are kept in a fresh medium. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. ANUSHA NADIKATLA

39. Myeloma cells have been genetically engineered (HGPRT-) such that they cannot use Hypoxanthine, Aminopterin, and Thymidine (HAT medium) as a source for nucleic acid biosynthesis and will die in culture. These cells are composed of a mixture of hybridomas (fused cells), free myeloma cells and free lymphocytes. Only B cells that have fused with the engineered myeloma cells will survive in culture when grown in HAT medium. ANUSHA NADIKATLA

40. STEP-5: ALLOW UNFUSED B CELLS TO DIE. ADD HAT CULTURE TO KILL UNFUSED MYELOMA CELLS Cells are plated in hypoxanthine-aminopterin-thymidine (HAT) selection medium – inhibitor of aminoterin which blocks nucleotide synthesis. Only fused cells with grow on HAT. Cells are distributed on feeder cells (murine bone-marrow ) to promote growth of the hybridomal cells. This happens in 7-10 days of culture. Selection of a single antibody producing hybrid cells is very important. This is possible if the hybridomas are isolated and grown individually. ANUSHA NADIKATLA

42. The suspension of hybridoma cells is so diluted that the individual aliquots contain on an average one cell each. These cells, when grown in a regular culture medium, produce the desired antibody. Unfused myeloma cells die because they cannot use the salvage pathway to make nucleotides and they are poisoned with aminopterin that blocks their pathway to nucleotide synthesis. Fused myeloma cells and normal cells do not die because the normal cell partner can make nucleotides in the presence of aminopterin, it can use the salvage pathway. ANUSHA NADIKATLA

43. STEP-6: CLONING OF HYBRIDOMA CELL LINES BY “LIMITING DILUTION” OR EXPANSION & STABILISATION OF CLONES BY ASCITES PRODUCTION A mouse is inoculated with the cell and thereby becomes a factory for producing the mAb. Small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time. The hybridomas now are ready to be diluted and grown, thus obtaining a number of different colonies, each producing only one type of antibody. ANUSHA NADIKATLA

44. Single Hybridoma Clone by Limiting Dilution (getting one clone or less per well) ANUSHA NADIKATLA

45. STEP-7: SCREENING SUPERNATANT OF EACH CLONE FOR PRESENCE OF THE DESIRED ANTIBODY (ELISA) The hybridomas must be screened for the secretion of the antibody of desired specificity. The culture medium from each hybridoma culture is periodically tested for the desired antibody specificity. The two techniques namely ELISA and RIA are commonly used for this purpose. In both the assays, the antibody binds to the specific antigen (usually coated to plastic plates) and the unbound antibody and other components of the medium can be washed off. Thus, the hybridoma cells producing the desired antibody can be identified by screening. The antibody secreted by the hybrid cells is referred to as monoclonal antibody. ANUSHA NADIKATLA

46. ELISA PLATE ANUSHA NADIKATLA

47. STEP-8: GROW THE CHOSEN CLONE OF CELLS IN TISSUE CULTURE INDEFINITELY This is done in vitro on culture bottles. The single hybrid cells producing the desired antibody are isolated and cloned. Two techniques are commonly employed for cloning hybrid cells-limiting dilution method and soft agar method. Limiting dilution method: In this procedure, the suspension of hybridoma cells is serially diluted and the aliquots of each dilution are put into micro culture wells. The dilutions are so made that each aliquot in a well contains only a single hybrid cell. This ensures that the antibody produced is monoclonal. Soft agar method: In this technique, the hybridoma cells are cultured in soft agar. It is possible to simultaneously grow many cells in semisolid medium to form colonies. These colonies will be monoclonal in nature. In actual practice, both the above techniques are combined and used for maximal production of MAbs. ANUSHA NADIKATLA

48. STEP-9: HARVEST ANTIBODY FROM THE CULTURE SUPERNATANT The monoclonal antibody has to be subjected to biochemical and biophysical characterization for the desired specificity. It is also important to elucidate the MAb for the immunoglobulin class or sub-class, the epitope for which it is specific and the number of binding sites it possesses. The stability of the cell lines and the MAbs are important. The cells (and MAbs) must be characterized for their ability to withstand freezing, and thawing. The desired cell lines are frozen in liquid nitrogen at several stages of cloning and culture. STEP-10: CHARACTERIZATION AND STORAGE ANUSHA NADIKATLA

49. PROPAGATION OF MONOCLONAL ANTIBODY ANUSHA NADIKATLA

50. IN VITRO PROPAGATION OF mAb A major advantage of using mAB rather than polyclonal antiserum is the potential availability of almost infinite quantities of a specific monoclonal antibody toward a single epitope. In general, mAB is found either in the medium supporting the growth of a hybridoma in vitro or in ascitic fluid from a mouse inoculated with the hybridoma. BATCH TISSUE –CULTURE METHOD: The simplest approach for producimg mAB be in vitro is to grow the hybridoma cultures in batches and purify the mAB from the culture medium. Fetal bovine serum is used in most tissue- culture media and contains bovine immunoglobulin at about 50µg/ml. In most cases, hybridoma growing in 10% fetal calf serum(FCS) can be adapted with in 8 – 12 days to grow in < 1% FCS or in FCS free media. By this approach it yields concentrations that are typically below 20µg/ml. ANUSHA NADIKATLA

51. SEMI PERMEABLE –MEMBRANE –BASED SYSTEMS: In this method the use of a barrier, either a hollow fiber or a membrane, with a LMW(10,000-30,000KD),has been implemented in several devices to permit cells to grow at high densities. These devices are called semipermeable-membrane-based systems. In this method isolate the cells and mAB produced in a small chamber separated by a barrier from a larger compartment that contains the culture media. Two membrane- based systems are available: The mini-PREM(unisyn tech,Hopkinton) The CELLine(Integra Bioscience) ANUSHA NADIKATLA

52. ADVANTAGES:  Reduce the use of mice at the antibody-production stage.  It is a method of choice for large scale production.  Avoid the need to submit animal protocols to IACUC’s.  Decrease the need for laboratory personnel.  Using semi permeable membrane based systems produce mAB in high concentrations. DISADVANTAGES: Some hybridomas do not grow well in culture or are lost in culture. The loss of proper glycosylation of the antibody might make the antibody product unsuitable for in vivo experiments because of: Increased immunogenicity, reduced binding affinity. Changes in biologic functions, accelerated clearance in vivo. In vitro culture methods are generally more expensive and limited by the amount of equipment. Batch culture supernatants contain less mAB per ml of medium than the mouse ascites fluid. Membrane based in vitro methods are contaminated with dead hybridoma cells and dead hybridoma cell products, thus requiring early and expensive purification. ANUSHA NADIKATLA

53. IN VIVO PROPAGATION OF mAb MOUSE ASCITES METHOD In vivo propagation involves ascites production from rodent hybridomas. During this process,the monoclonal cells are injected into the peritoneal cavity of a mouse and allowed to grow. After 2 weeks,there is abuild- up of ascitic fluid, which contains the antibodies. These antibodies can then be removed from the peritoneal cavity using a needle syringe. Advantages: This method usually produces very high concentrations that often do not require further conc. Procedures that can denature antibody and decrease effectiveness. The high conc. of the desired mAB in this method avoids the effects of contaminants. Relatively inexpensive and easy Disadvantages: This method involves the continued use of mice requiring daily observation. mAB produced by this method can contain various mouse proteins and contaminants. This method can cause significant pain or distress in mice. There are ethical concerns with using animals. ANUSHA NADIKATLA

55. HARVESTING mAb BY AFFINITY CHROMATOGRAPHY- ANTIBODY PURIFICATION Antigen can be bound to the support matrix in order to purify antigen-specific antibody from a polyclonal antiserum. ANUSHA NADIKATLA

56. LARGE SCALE PRODUCTION OF mAb The production MAbs in the culture bottles is rather low (5-10 (ig/ml). The yield can be increased by growing the hybrid cells as ascites in the peritoneal cavity of mice. The ascitic fluid contains about 5-20 mg of MAb/ml. This is far superior than the in vitro cultivation techniques. But collection of MAb from ascitic fluid is associated with the heavy risk of contamination by pathogenic organisms of the animal. In addition, several animals have to be sacrificed to produce MAb. Hence, many workers prefer in vitro techniques rather than the use of animals. ANUSHA NADIKATLA

57. EVALUATION OF MAB 1. Transchelation Challenge Test: The samples were submitted to two different challenge media, serum and cysteine solution, in order to test the "in vitro stability". Serum: 10 µg of the labelled antibody is added to 1 mL of fresh human serum. After a mild shaking, the sample was analysed by paper and ITLC chromatography, aswell as electrophoresis if considered necessary. Cysteine: Two cysteine solutions were added to a solution of the radiolabelled antibody, such that the final molar ratio were 0.5:1 and 500:1 in relation to MoAb. The protein concentration was 350 µg/mL. After incubations at room temperature and 37°C for over 4 hrs the solutions were analysed by a paper chromatography system, whose mobile phase was PBS ,pH 7.2. ANUSHA NADIKATLA

58. 2. Immunoreactivity studies: a. Micro ELISA system: The immunoreactivity of reduced and labelled was determined in a competitive binding assay against the native antibody by a micro ELISA system, Polystyrene plates (high binding, COSTAR), were coated with ior-CEA-1 diluted in coating buffer. The plate was incubated for 18 h at 37°C and then washed with washing buffer (phosphate buffer saline (PBS) with 0.05% of Tween 20) three times with 200 µL of buffer per well. Two hundred µl of CEA (2 µg/mL) and modified or control antibody were incubated in Eppendorf vials to a final volume at 200 µL and vortexed for 10 sec. After incubation at 37°C for 1 h the plate was washed and a solution of p-nitrophenyl phosphate in diethanolamine buffer. The color was developed in 30 min at room temperature and the reaction was stopped with NaOH 3 M . The absorbance values were measured in a ELISA plate reader (Organon, Teknica) at 405 nm. b. Animal biodistribution study: Female mice weighing 18-25 gm were used in the experiment. About 100 µL of labelled mAB , were injected intraperitoneally. Four hr later, the animals were sacrificed. Radioactivity in each organ was counted in a gamma counter and recorded as percentage of dose/g tissue. ANUSHA NADIKATLA

59. APPLICATIONS OF MAB The application of monoclonal antibodies can be broadly categorized as: DIAGNOSTIC APPLICATION CATALYTIC MAB (ABZYMES) THERAPEUTIC APPLICATION ANUSHA NADIKATLA

60. DIAGNOSTIC APPLICATION The western blot test & immuno dot blot tests detect the protein on a membrane. Useful in immunohistochemistry, which detect antigen in fixed tissue sections. Immuno fluoresence test,which detect the substance in a frogen tissue section or in live cells. MAbs are utilized in diagnostic kits for the diagnosis of various infectious diseases, detecting pregnancy, monitoring drug levels, matching histocompatibility antigen, detecting diabetes, cancer and in immunoscintigraphy. FDA licensed a new diagnostic imaging agent that can determine the extent of disease in patients diagnosed with small cell lung cancer (SCLC). Because these agents can detect tumor in different part of the body at one time, it can help physician to advice certain patients with advanced forms of the disease about treatment option without requiring further diagnostic tests. The new agent, Nofetumomab, is a fragment of a monoclonal antibody that when tagged with the radioisotope technique, can detect a protein found on the surface of most small lung cancer cells. ANUSHA NADIKATLA

61. CATALYTIC MAb (ABZYMES) Abzymes are usually raised in lab animals immunized against synthetic haptens, but some natural abzymes can be found in normal humans (anti-vasoactive intestinal peptide autoantibodies) and in patients with autoimmune diseases such as systemic lupus erythematosus, where they can bind to and hydrolyze DNA. The antibodies are extremely efficient at binding ground states of the target molecule while enzymes obtained their catalytic efficiency from tight binding of the transition state for the reaction. Thus antibodies can be made efficient catalysts if they are made for reaction transition state. ANUSHA NADIKATLA

62. IMMUNOCONJUGATE These are antibodies conjugated (joined) to a second molecule, usually a toxin, radioisotope or label. For mAB’s targeted drug delivery, a drug is bound covalently to an antibody. The resulting immunoconjugate may contain a spacer between drug and antibody (or) a polymer to increase the number of drug molecules that can be bound to each antibody. The drug can be incorporated noncovalently into a liposome or microsphere to which the targeting antibody is bound to surface is known as Immunoliposome or Immunomicrosphere. ANUSHA NADIKATLA

63. THERAPEUTIC APPLICATION Improving the outcome of bone marrow transplantation by using CD52 MAbs to prevent Graft-Versus-Host disease and Graft rejection. Graft-versus-Host Disease (GVHD) is a major cause of mortality and mobidity after allogenic bone marrow transplantation, but can be avoided by removing T- lymphocytes from the donor bone marrow. However, T-cell depletion increases the risk of graft rejection. This study examined the use of CD52 MAb to eliminate T- cells from both donor marrow and recipient to prevent both GVHD and rejection. Alemtuzumab is the monoclonal antibody used for this purpose. ANUSHA NADIKATLA

64. mAb IN TISSUE TRANSPLANTATION Muromonoab-CD3: Muromonoab-CD3 is used for the treatment of acute organ transplant rejection. It is effective in preventing graft rejection after kidney, heart or livertransplantation. Muromonoab-CD3 is effective in patients who after acute cardiac or liver allograft rejection do not respond to steroid therapy. ANUSHA NADIKATLA

66. mAb IN PSORIASIS Psoriasis is a disease of the immune system that involves T lymphocytes. The etiology and pathogenesis of psoriasis results from complex communications that cause activation of T lymphocytes and trafficking to the skin. Further reactivation causes inflammation and overproduction of skin, resulting in lesions and plaques ANUSHA NADIKATLA

68. Efalizumab is a humanized IgG1_ antibody produced by recombinant DNA technology. It exhibits immunosuppressive function. It binds to CD11a, which is the α-subunit of leukocyte function antigen (LFA)-1. Efalizumab decreases the cell surface expression of CD11a, which is expressed on all leukocytes. ANUSHA NADIKATLA

69. mAb IN RHEUMATOID ARTHRITIS Monoclonal antibodies have been shown to be clinically effective in suppressing inflammation in RA. They may be used to treat disease flares and in combination with conventional DMARDs to achieve better disease control. some mAbs, such as anti-CD4,improve disease for a prolonged period in animal models of RA. ANUSHA NADIKATLA

70. It is a chimeric monoclonal antibody produced by recombinant DNA technology and is directed against TNF-α. It is composed of human constant and mouse variable regions. Infliximab binds to the soluble and the membrane bound form of TNFα, resulting in the neutralization of its biological activity. ANUSHA NADIKATLA

71. mAb IN THROMBOSIS Abciximab (ReoPro): Abciximab is a Fab fragment of a chimeric monoclonal antibody that is directed against GPIIb/IIIa receptors. These receptors are located on platelets where they are involved in platelet aggregation. Abciximab inhibits platelet aggregation by blocking GPIIb/IIIa receptors, thus preventing the binding of fibrinogen, vonWillebrand factor and other molecules promoting adhesion to the receptors on platelets. It increases bleeding and activated clotting times and reduces the response of platelets to adenosine diphosphate. ANUSHA NADIKATLA

72. mAb IN CANCER TREATMENT It involves mAB that bind only to cancer cell- specific antigens and induce an immunological response against the target cancer cell. The mAB can also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate. ANUSHA NADIKATLA

73. MONOCLONAL ANTIBODIES FOR CANCER Radio immunotherapy Antibody-directed enzyme prodrug therapy (ADEPT) Immunoliposomes ADEPT(antibody directed enzyme prodrug therapy) ADCC(antibody dependent cell-mediated cytotoxicity) CDC(complement dependent cytotoxicity) scFV(single-chain Fv fragment) ANUSHA NADIKATLA

75. EXAMPLES OF FDAAPPROVED TREATMENTS OF CERTAIN CANCERS MAB NAME TRADE NAME USED TO TREAT APPROVED IN Rituximab Rituxan Non-Hodglymphoma 1997 Trastuzumab Herceptin Breast cancer 1998 Gemtuzumab ozogamicin* Mylotarg Acute myelogenous leukemia (AML) 2000 Alemtuzumab Campath Chronic lymphocytic leukemia (CLL) 2001 Ibritumomab tiuxetan* Zevalin Non-Hodgkin lymphoma 2002 Tositumomab* Bexxar Non-Hodgkin lymphoma 2003 Cetuximab Erbitux Colorectal cancer Head & neck cancers 2004 2006 Bevacizumab Avastin Colorectal cancer 2004 ANUSHA NADIKATLA

76. mAb IN AUTO IMMUNE DISEASES Monoclonal antibodies used for autoimmune diseases , which are effective in rheumatoid arthritis, crohn’s disease and ulcerative colitis.(Infliximab,Limumab) Preventing acute rejection of kidney transplants (Basiliximab,Daclizumab). Useful in moderate to severe allergic asthma(Omalizumab). Immunoliposomes are used for intracellular delivery of compounds that intrinsically do not enter the diseased cells. • Eg: Methotrexate-γ-aspartate. ANUSHA NADIKATLA

77. CLINICALAPPLICATIONS OF MONOCLONALS IN AUTOIMMUNITYAND TRANSPLANTATION MEDICINE Zenapax Targeted to IL-2 Receptor alpha subunit on activated T-Cells (Anti-TAC) Modulates acute kidney rejection Orthoclone OKT3 Targeted to CD3 co-receptor on activated T-cells Controls rejection in liver, heart, and kidney transplants Remicade Targets Tumor Necrosis Factor mediator of inflammation Treatment of Autoimmune Rheumatoid Arthritis and Crohn’s Disease Xolair Antibody to IgE (Antibody to an antibody) Type 1 Allergy Treatment ANUSHA NADIKATLA

78. mAb IN ALLERGY Allergic disorders, including asthma, allergic rhino conjunctivitis, atopic dermatitis, food allergies, urticaria and anaphylaxis have significant impacts on our daily lives. ANUSHA NADIKATLA

79. TYPE TARGET MODE Omalizumab IgE Humanized Pascolizumab IL-4 Humanized Certolizumab TNF-α receptor Humanized Rituximab CD20 CD20 Chimeric ANUSHA NADIKATLA

81. PROBLEMS ASSOCIATED WITH MOUSE mAb The therapeutic use of rodent monoclonal antibodies in humans is limited by their immunogenic, short circulating half-life, and inability to efficiently trigger human effectors mechanisms.This is due to differences between the mouse and humans. Also severe allergic response in human when mouse mAb are introduced to a patient. Also constant region of marine mAb are not effective in interacting with human effectors molecules. Main difficulty is that mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti-mouse antibodies") This problem derives from the fact that although antibodies show some conservation there are many sequence differences between rodent antibodies and human antibodies ANUSHA NADIKATLA

82. PROBLEMS ASSOCIATED WITH mAb’s Cancer cells are heterogenous, so those cells that are not recognised by MoAb can escape and proliferate. Some tumours contain semidead cores with poor circulation and thus cannot be reached by monoclonals. MoAb’s can interact with circulating target antigens before reaching this target. Patients can experience possible immunogenic reactions. A MoAb is not as specific invivo as would be predicted from invitro studies.i.e, tumour antibodies may bind to normal cells as well as target cells. MoAb’s have poor penetration incase of solid tumors due to lack of vasculature. For these reasons, it is proven more effective to combine MoAb with standard chemotherapeutic agents. ANUSHA NADIKATLA

83. CONCLUSION The monoclonal antibody production technology has revolutionized the world of Biotechnology. Advances in genetic engineering over the years have provided numerous ways to design mAbs that are more robust and efficacious compared with their original murine version. mAbs have not only been used as diagnostics, therapeutics, research reagents, drug targettor for various infectious diseases but also cancerous, metabolic and hormonal disorders. mAb technology in conjunction with recombinant DNA technology has successfully led to the reconstruction of chimeric, humanized and fully human antibodies and has enormous potentials for therapeutic uses. ANUSHA NADIKATLA

84. REFERENCES 1. Anon. (1989). Code of Practice for the Production of Monoclonal Antibodies, 6 pp. Rijswijk, The Netherlands: Veterinary Public Health Inspectorate, Department of Animal Experimentation. 2. Kuhlmann, I., Kurth, W. & Ruhdel, I. (1989). Monoclonal antibodies: in vivo and in vitro production on a laboratory scale, with consideration of the legal aspects of animal protection. ATLA 17, 73ñ82. 3. van der Kamp, M. & de Leeuw, W.A. (1996). Short review of in vitro production methods for monoclonal antibodies. NCA Newsletter 3, 10ñ12. 4. Hendriksen, C., Rozing, J., van der Kamp, M. & de Leeuw, W. (1996). The production of monoclonal antibodies: are animals still needed? ATLA 24, 109ñ110. 5. “Production of Monoclonal Antibodies” ;Wayne M. Yokoyama, Michelle Christensen, Gary Dos Santos,Diane Miller, Jason Ho, Tao, Wu, Michael Dziegelewski, Francisca A. Neethling ; Current Protocols in immunology, unit 2.5 6. “Production of monoclonal antibodies: Strategy and tactics “; St. Groth, Doris Scheidegger ; Journal of Immunological Methods, Volume 35, Issues 1–2, 15 July 1980, Pages 1-21 7. Bruno CJ, Jacobson JM, Ibalizumab: an anti-CD4 monoclonal antibody for the treatment of HIV-1 infection; J Antimicrob Chemother;1839-41; 2010 8. “Monoclonal Antibody Production” , NATIONAL ACADEMY PRESS Washington, DC 1999 9. Bruce A. Chamber, Jeol Neal et.al, Targeted therapies, Tyrosine kinase inhibitors, monoclonal antibodies; Goodman and Gilmans, Pharmacological basis of therapeutics; 12;1731-50; 2010. ANUSHA NADIKATLA

MONOCLONAL ANTIBODIES ( MAB )

N Anusha

MONOCLONAL ANTIBODIES ( MAB )