MONOCLONAL ANTIBODIES A Project Report For Elective Subject Submitted To Hemchandracharya North Gujarat University, Patan.

Transcription

1 MONOCLONAL ANTIBODIES A Project Report For Elective Subject Submitted To Hemchandracharya North Gujarat University, Patan. In Partial Fulfillment of the Requirement for the Degree Certificate of Bachelor of Pharmacy Year: Submitted By, HETAL R. PARMAR Shree S.K.Patel College of Pharmaceutical Education & Research, Ganpat Vidyanagar, Kherva

2 CERTIFICATE This is to certify that the project report for the elective subject entitled monoclonal antibodies is the bonafide work of HETAL R. PARMAR satisfactorily carried under my guidance and supervision in the Pharmacology Department of Shree S.K.Patel College of Pharmaceutical Education and Research, Ganpat Vidyanagar, during the academic year This work is upto my satisfaction. Guide: Dr.N.J.PATEL M.Pharm, Ph.D. Professer& H.O.D. of Pharmacology, Shree S.K. Patel College of Pharmaceutical Education & Research, Ganpat Vidyanagar. Principal: Dr.M.M.Patel M.Pharm, Ph.D, LLB, FIC. Department of Pharmaceutical Technology Shree S.K. Patel College of Pharmaceutical Education & Research, Ganpat Vidyanagar. Date: Place:

3 ACKNOWLEDGEMENT The successful completion of a project MONOCLONAL ANTIBODIES is generally not an individual effort. It is an outcome of the cumulative effort of a number of persons, each having his own importance to the objective. This session is a vote of thanks and gratitude towards all those persons who have directly or indirectly contributed in their own special way towards the completion of this project. First of all I would like to thank my guide respected Dr.N.J.Patel for giving me guidance and also for supporting me in all the way to complete this project. I also like to thank our respected faculty members R.J.Patel, R.K.Patel, N.J.PATEL, P.V.Gohil, for their valued guidance and encouragement towards the completion of our project. I am also very much thankful to our honorable principal Dr.M.M.Patel for providing infrastructure and research facilities at college. And I am really grateful to all those, whose ideas have been helpful in preparing this report. I am also thankful to our librarian Mr.P.I.Patel, Mahadevbhai, Mukeshbhai for providing me books & reference. I heartily thankful to Chaula madam for her great help to me in computer laboratory. I am thankful to all my friends and specially NILAM and my brother JIGAR for always help me in my all diffuculty. At last but not at least I express thanks to my family members for their support and constant encouragement for successful completion of my project work. Ganpat Vidyanagar Hetal R. Parmar Final B.Pharm.

4 Dedicated to God, My Family & Teachers

5

6 CONTENT INDEX 1. INTRODUCTION 1 2. MAb DEVELOPMENT STAGES 5 3. PRODUCTION 8 COMMERCIAL PRODUCTION OF MONOCLONAL ANTIBODIES 4. APPLICATION OF MONOCLONAL ANTIBODIES 17 CLINICAL APPLICATION OF MONOCLONAL ANTIBODIES 5. USES OF MONOCLONAL ANTIBODIES ADVANTAGES OF MONOCLONAL ANTIBODIES LIMITATION OF MONOCLONAL ANTIBODIES REFERENCES 31 PAGE NO

7 INTRODUCTION Monoclonal antibodies Monoclonal antibodies are proteins produced in the laboratory from a single clone of a B-cell, the type of cells of the immune system that make antibodies. Antibodies, also known as immunoglobulins (Igs), are proteins that help identify foreign substances to the immune system, such as a bacteria or a virus. Antibodies work by binding to the foreign substance to mark it as foreign. The substance that the antibody binds to is called an antigen. All monoclonal antibodies of a particular type bind to the same antigen, which distinguishes them from polyclonal antibodies. The structure of most antibodies can be divided into two parts: the section that binds the antigen and a section that identifies the type of antibody. This second region is called a constant region, because it is essentially the same within the same type of antibody. The most common type of antibody is IgG (immunoglobulin gamma), which is found in the blood and body fluids. For cancer treatments, monoclonal antibodies are often humanized. This involves using human sequences for the constant regions and using mouse or other animalderived sequence for the binding region. Humanization reduces the immune reaction of the patient to the antibody itself. A final strategy involves special antibodies that are linked (conjugated) to a substance that is deadly to the cancer cells. Both radioactive isotopes, like yttrium 90, and toxins produced by bacteria, like pseudomonas exotoxin, have been successfully conjugated to antibodies. The antibodies are then used to specifically destroy the tumor cells with the radioactivity or toxic substance. The use of monoclonal antibodies is a useful approach to cancer therapy and as scientists learn more about the function of the immune system and cancer, new antibodies and new strategies promise to become more and more effective. 1 SKPCPER

8 Antibody proteins that bind to a specific target molecule (antigen) at one specific site (antigenic site). In response to either infection of immunization with a foreign agent, the immune system generates many different antibodies that bind to the foreign molecules. Individual antibodies within this polyclonal antibody pool bind to specific sites on a target molecule known as epitopes. Isolation of an individual antibody within the polyclonal antibody pool would allow biochemical and biological characterization of a highly specific molecular entity targeting only a single epitope. Realization of the therapeutic potential of such specificity launched research into the development of methods to isolate and continuously generate a supply of a single lineage of antibody, a monoclonal antibody (mab). In 1974, W. Köhler and C. Milstein developed a process for the generation of monoclonal antibodies. In their process, fusion of an individual B cell (or B lymphocyte), which produces an antibody with a single specificity but has a finite life span, with a myeloma (B cell tumor) cell, which can be grown indefinitely in culture, results in a hybridoma cell. Generation of monoclonal antibodies through the hybridoma process worked well with B cells from rodents but not with B cells from humans. Consequently, the majority of the first monoclonal antibodies were from mice. When administered into humans as therapeutic agents in experimental tests, the human immune system recognized the mouse monoclonal antibodies as foreign agents, causing an immune response, which was sometimes severe. Although encouraging improvements in disease were sometimes seen, this response made murine (mouse) antibodies unacceptable for use in humans with a functional immune system.. In most mammals, each antibody is composed of two different polypeptides, the immunoglobulin heavy chain (IgH) and the immunoglobulin light chain (IgL). Comparison of the protein sequences of either heavy of light antibody chain reveals a portion that typically varies from one antibody to the next, the variable region, and a portion that is conserved, the constant region. A heavy and a light 2 SKPCPER

9 chain are folded together in an antibody to align their respective variable and constant regions. The unique shape of the cofolded heavy- and light-chain variable domains creates the variable domain of the antibody, which fits around the shape of the target epitope and confers the binding specificity of the antibody. Disease areas that currently are especially amenable to antibody-based treatments include cancer, immune dysregulation, and infection. Depending upon the disease and the biology of the target, therapeutic monoclonal antibodies can have different mechanisms of action. A therapeutic monoclonal antibody may bind and neutralize the normal function of a target. For example, a monoclonal antibody that blocks the activity of the protein needed for the survival of a cancer cell causes the cell's death. Another therapeutic monoclonal antibody may bind and activate the normal function of a target. Any of a class of highly specific antibodies produced by the clones of a single hybrid cell formed in the laboratory by the fusion of a B cell with a tumor cell and widely used in medical and biological research. The human immune system is complex, but one of the most easily accessible parts is a group of chemicals called antibodies that circulate in the bloodstream. Antibodies are the key to the success of vaccination. Injection of a vaccine stimulates a kind of white blood cell (lymphocyte) called a B cell to produce antibodies against proteins in the vaccine. Such antibodies home in on specific foreign proteins so that they can be destroyed. Vaccination confers long-lasting immunity because the B cells that have been stimulated to produce the antibodies reproduce, and the B-cell line remains in the bloodstream, prepared to produce antibodies against a reinvasion of the specific protein. The same process, of course, happens naturally in response to infection. The key to antibody action is that a line of antibodies that bind to one kind of protein or other molecule -- called the antigen -- is very specific. Normally, antibodies in a given line do not bind to any molecules except that antigen, 3 SKPCPER

10 although sometimes antibodies can be fooled by a protein that closely resembles the antigen in some way; this can result in autoimmune diseases such as type I diabetes or rheumatoid arthritis. In 1975 César Milstein and Georges Köhler developed in England a method of producing large amounts of antibodies specific to a given antigen by a cloning are used to pick out a hybridoma that produces a specific desired antibody; this cell is separated from the others and encouraged to reproduce in large quantities. Antibodies from such a cell line are called monoclonal because they are produced from the clones of a single hybridoma. All of the monoclonal antibodies from a single B cell react with the chosen antigen. Just like natural antibodies, monoclonal antibodies track down proteins or other chemicals with exquisite specificity. Thus, they can be used in diagnosis of such diseases as muscular dystrophy, one of the first applications. By combining a monoclonal antibody with a poison, cells that have a given protein on their surface can be tracked down by the antibody and destroyed. This has been successful against some types of cancer, especially certain breast cancers and leukemias. 4 SKPCPER

11 MAb Development Stages Stages of hybridoma development 1. Immunisation 4 Bale/C female mice will be immunized with a series of injections containing the mixture of the immunogen and Freund s adjuvant. The first intraperitoneal injection will consist of 0,1ml protein in PBS mixed in 1/1 ratio with 0,1ml of complete Freund s adjuvant. The other series of injections with incomplete Freund s adjuvant will replace the complete one. Preimmune serum is taken on day 0. Sera will be collected during the protocol and titers will be determined by ELISA in order to select the best responding mouse after. If the antibody titer is too low after the last injection, you may request additional injections. If the ELISA test remains negative, the immunisation should be terminated because the probability of success is too low. 2. Development of screening method for antibody detection Antigen will be coated to microtiter plate using Covalab technologies. This procedure is often used for the screening of antiserum. The remaining free sites on the solid support will be blocked in order to avoid the non-specific interaction of the antiserum for the ELISA test; several parameters will be optimized such as: antigen concentration, serum dilutions, buffers and solvent, incubation time, temperature of the immunoreactions, dilutions of anti-mouse peroxydase conjugate. The customer will control the immunoreactivity of the antiserum. 5 SKPCPER

12 3. Fusion and screening Final boost: At 3 to 4 days before the fusion, the mouse exhibiting the highest antibody titer will be injected with antigen as a final boost. This will allow most of the circulating antibodies to be cleared from the blood stream by the mouse. Fusion of murine myeloma cell line (Sp2/O-Ag-14) and spleen cells from the best responding animal using PEG (polyethylene glycol) The HAT mixture of drugs will be used to select against the growth of the myeloma cells. The cells will then be dispensed in 96-well microtiter plates and placed at 37 C in a CO2 incubator. 5 to 7 days after, cells are fed by adding 1/2 volume of HAT medium to each well. Visual control is made over 3 weeks for growing colonies and these will be identified for antibody secretion. Screening: The supernatants from the wells containing hybridomas will be removed and the immunoreactivity of the antibodies is assessed by ELISA Amplification of positive colonies: three vials of every hybridoma will be stored in liquid nitrogen. 4. Cloning and isotyping After confirmation of positive hybridomas, the next step will be the cloning of antibody-producing cells. The original positive well often contains more than one clone of hybridoma cells and some have an unstable assortment of chromosomes. Single cell cloning ensures that cells that produce the antibody of interest are monoclonal and that the secretion of this antibody can be maintained in a stable way. Different approaches to cloning are used but limiting dilution performs usually cloning. Within three weeks of cloning, supernatants from wells containing hybridomas will be screened to determine the clone that produces the desired antibody. Positive wells that are likely to be of monoclonal origin will be expanded. Class and subclass isotyping of all selected clones will be determined. Remark: Cloning and isotyping of 3 hybridomas selected at the previous step, 6 SKPCPER

13 freezing of hybridomas secreting specific immunoglobulins at clonal stage: 3 hybridomas at maximum, 5 vials/hybridoma, 3 different freezing dates. Cloning by limiting dilution and expansion of cells Screening and amplification of positive clones Isotyping of clones and determination of Ig subclass Storage of selected hybridomas in liquid nitrogen The final clones are sent to the customer for testing and confirmation 7 SKPCPER

14 Production If a foreign substance (an antigen) is injected into a vertebrate such as a mouse or a human, some of the immune system's B-cells will turn into plasma cells and start to produce antibodies that bind to that antigen. Each B-cell produces only one kind of antibody, but different B-cells will produce structurally different antibodies that bind to different parts ("epitopes") of the antigen. This natural mixture of antibodies is known as polyclonal antibodies. To produce monoclonal antibodies, one removes B-cells from the spleen of an animal that has been challenged with the antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B- cell cancer). This fusion is done by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of antibodies. The hybridomas are sufficiently diluted and grown, so that one obtains a number of different colonies, each producing only one type of antibody. The antibodies from the different colonies are then tested for their ability to bind to the antigen (for example with a test such as ELISA), and the most effective one is picked out. Monoclonal antibodies can be produced in cell culture or in animals. When the hybridoma cells are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an antibody-rich fluid called ascites fluid. In the above process, one uses myeloma cell lines that have lost their ability to produce their own antibodies, so as to not dilute the target antibody. Furthermore, one employs only myeloma cells that have lost a specific enzyme (hypoxanthine-guanine phosphoribosyltransferase, HGPRT) and therefore cannot grow under certain conditions (namely in the presence of HAT medium). Fusions between healthy B-cells and myeloma cells are rare, but when one 8 SKPCPER

15 succeeds, then the healthy partner supplies the needed enzyme and the fused cell can survive in HAT medium. Monoclonal Antibody Production Fusing single antibody-forming cells to tumor cells grown in culture produces monoclonal antibodies. The resulting cell is called a hybridoma. Each hybridoma produces relatively large quantities of identical antibody molecules. By allowing the hybridoma to multiply in culture, it is possible to produce a population of cells, each of which produces identical antibody molecules. These antibodies are called "monoclonal antibodies" 9 SKPCPER

16 Four basis steps are involved in the production of a given monoclonal antibody: 1. Immunization 2. Generation of B cell hybridomas by fusing primed B cells and mycelia cells 3. Selection and the screening of the resulting clones for those that secrete antibody with the desired specificity. 4. Cloning by propagating the desired hybridomas. Immunization Immunization of animals with immunogens is performed by injecting microgram or milligram quantities of immunogen mixed with an adjuvant (aluminium salts, Freund s complete or incomplete adjuvant), intradermally or subcutaneous at different times. The serum of animal is assayed for the relative concentration of antibodies of desired specificity at various time intervals. When the concentration of antibodies is found to be nearly optimal, the animal is sacrificed and the spleen, which contains a large number of plasma cells, is dissociated into single splenocytes by mechanical and/or enzymatic method. Cell fusion Splenocytes are then mixed with plamocytoma cells in an appropriate medium. This mixture is exposed to a high concentration (e.g.50%) of PEG for short time and fusion is allowed to proceed over a period of time. Mouse has been used in the production of monoclonal antibody secreting hybridoma with splenocytes; because these animals are inexpensive and several billion cells can be obtain from a mouse spleen. In contrast, mice 10 SKPCPER

17 are rarely used for the production of polyclonal antisera, because of their very low blood volume (=2 ml). The use of HGPRT cells (that cannot growin HAT medium) assured that only hybridomas (hybrid myeloma-spleen cells) are selected. After 7-10 days of culture in the HAT medium most of the wells contain dead cells, but few wells contain small clusters of viable cells. Each clusters represents clonal expansion of a hybridoma. After HAT selection, single cells are transferred and cultured in separate wells to ensure the monoclonality of the secreted antibody. Wells containing viable clusters are then screened for antibody production and antibody positive clones are subcultured at low cell densities, again to ensure clonal purity in each microwell. The first hybridoma obtained by Kohler and Milstein secreted not only antibody from the spleen B cell but also unwanted antibody from the myloma cell as well as one hybrid antibody containing heavy or light chains from both original parent cells. To avoid this difficulty a HGRPT, Ab myeloma cell was chosen as idea fusion partner. This fusion partner has the immortal properties of a cancer cell but does not secrete its own antibody or gene product. Hybridomas generated with this fusion partner thus secrete only the antibody from the B cell partner. These hybridomas can be propagated in tissue culture to give rise to large clones secreting homogeneous monoclonal antibody. Selection and screening of the clones for monoclonal antibody specificity Once pure clones of antibody-secreting hybridomas are obtained, they must be screened for the desired antibody specificity. Screening of hybirdomas for secretion of antibodies of desired specificity usually follows selection of hybridoma cells in HAT medium. After fusion, the cells may be transferred to HAT medium in tissue culture flasks. After an appropriate incubation time, all 11 SKPCPER

18 viable cells will be hybridomas. They are removed from the flasks, tranferred to regular culture medium and aliquots are distributed among the well of 96-well plastic culture plates. The supernatant of each hybridoma culture can be assayed for particular antigen specificity in various ways. Two methods are frequently used (a) ELISA and (b) RIA.Both are easily adapted to mass screening with 96- well plastic culture plates. In both assays, antigen that reacts with the desired antibody is bound to the bottom of 96-well plates, and washed to remove unbound antigen. Supernant from each hybridoma well is added to separate well and incubated for an appropriate period of time. If a simple of supernatant contains the desired antibody it will bind to antigen and will remain associated with the well as unbouned material is washed off. In ELISA, this antibody is then detected by an immunoconjugate cosisting of two component covalently linked to each other. One component is an antibody specific for an epitope in the constant domain of the first antibody (e.g. mouse Fc or mouse Y chain). The second component is an enzyme such as alkaline phosphatase or horseradish peroxidase. After another washing step, a colorless substrate that is covered to a colored product by the enzyme of the immunoconjugate is added. After an appropriate incubation, the enzymatic reaction is stopped and the optical density of each well is determined at the respective gamma max.with a specialized colorimeter known as a 'plate reader. In RIA the anti-isotype antibody is radio labeled; bound label can be detected by counting the wells individually in a gamma counter or the entire plate can be exposed to X- ray film. If the desired monoclonal antibody is specific for a cell-membrane molecule, immunofluorescent techniques can be used for screening. In this case, target cells with the particular cells membrane antigen are stained with monoclonal antibody In micro liter well and visualized by the addition of fluorochrome-conjugated antiisotype antibody. 12 SKPCPER

19 Cloning of hybridomas secreting specific monoclonal antibody Single cells secreting the desired antibody are isolated from positive cultures and propagated into cell lines. There are two cloning techniques, which are most widely used: 1.Liminting dilution 2. Soft agar. In limiting dilution, the cells in the culture are enumerated, diluted and aliquoted into new wells so that only one cell found in well. Cells are allowed to regrow and the procedure is repeated several times to increase the probability that all the coils in a given well are monoclonal. The second method is based on the fact that many malignant cells will proliferate, forming spherical colonies, in a semisolid medium containing low amount of agar. If the culture can be reliably dispersed into single cells and the single cells and the cell concentration is such that the colonies will be well spaced, then visible colonies picked out of the agar are likely to be monoclonal. When a hybridoma is grown in tissue culture flasks, the antibody is secreted into the medium at fairly low concentration ( micrograms/ml). An increased in the yield of monoclonal antibody secreting hybridoma can also be propagated in the peritoneal cavity of histocomplatible mice, where it secretes the monoclonal antibody into the ascites fluid at concentration of 1-25mg/ml. The antibodies can be purified from the mouse ascites fluid by chromatography. To meet the increased demand of monoclonal antibodies, biotechnology companies have been developing various techniques to increased yield. Demon Biotech Company encapsulates hybridomas in alginate gels, which allow nutrients to flow in, and waste products and antibodies to flow out. In these capsules much higher concentration of hybridomas cells can be achieved than in 13 SKPCPER

20 tissue culture, as a result 100 fold greater yield of antibody production has been obtained. Another approach has been used by cell tech in UK.In this method hybridomas are grown in 100-liter fermenters, which yield 100 gram of monoclonal antibodies in a 2-week period. Commercial production of monoclonal antibodies Commercially, monoclonal antibodies are produced by two methods: 1. Ascites production in mice, 2. Invitro fermentation. Ascites production in mice In this method hybridomas cells are injected into the peritoneal cavity of histocomapatible mice. The mice are pretreated by i.v injection of pristine to irritate the peritoneal cavity and to establish a conditioned environment that facilitates the growth of ascetics tumor. The fluid produced can contain a high concentration of secreted monoclonal antibodies 3 to 15 mg\ ml, and 3 to 5 ml or more can be harvested per mouse. Many pharmaceutical grade monoaclonal antibodies have been produced using this technique especially for initial clinical trials. The first monoclonal antibodies approved by FDA for therapeutic use OKTS is produced by ascites. The methods has the following drawbacks: 1. It is very costly. 2. It is not reliable, product its contaminated with low levels of normal mouse inmmunoglobulin as well as other mouse protein. 3. Viruses can be introduced. 4. Human monoclonal antibodies are very difficult and expensive to produce as ascites in mice. 14 SKPCPER

21 5. The human hybridomas may require special immunodeficient mice 6. The antibody yield in ascites is of then lower than murine hybridomas. Invitro fermetation Fermentation is widely accepted method for the production of monoclonal antibodies because of the problems associated with ascites produced monoclonal antibodies. Potent steps of fermentation based manufacturing process are presented in table. This method has offered certain advantages. 1. There is no contamination with normal mouse immunoglobulin. 2. The process can be cost effective especially as serum requirements in the culture medium are reduced. 3. It is reliable. 4. It can be directly scaled up from small pilot bioreacters to very large production scale. 5. There is very less contamination by adventious agent. 6. Human monoclonal antibodies can be produce at lower level (0.1 to 0.5 mg per ml). The major problem in this method is the contamination of product with serum or protein based on growth factors and other constituents. 15 SKPCPER

22 Outline of potential steps used in fermentation based manufacturing 1. Preparation of cells banks a). Masier cells banks b). Manufacturers work cells banks (MWCB). 2. Fermentation a). Preculture of MWCB b). Fermentation c). Harvest of culture medium d). Clarification and concentration. 3. Downstream processing a). Initial purification b). Digestion of fragments (Pepsin, papain) c). Further purification. d). Conjugation (Radioisotopes chelator, toxin, drugs). e). Final purification 4. Pharmaceutical manufacturing a). Formulation b) Violating c) Lyophilization The stirred tank and airlift systems are used for the production of because they can be readily scaled up to very large size (> 1000L). The harvested culture broth containing the monoclonal antibody product requires the separation of cells and cell and debris. This clarification step can be accomplished by centrifugation or filtration. The product is then concentrated (10 to 20 fold) by ultra filtration to reduce volume prior to subsequent downstream processing. 16 SKPCPER

23 Application of monoclonal antibodies Monoclonal antibodies are having a remarkable range of applications. The following criteria are helpful to understand the areas of MAbs application. 1.Antibodies against a range of molecules including those on cell surface and microorganisms are possible. 2. The ability of antibodies to lyse target cells in the presence of compliment. 3. Hybridomas technology can produce a range of hybridomas cell line secreting antibodies against an antigenic determinant. 4.In affinity chromatography a powerful purification technique wherein antibody bound in solid systems are used to separate specific antigen from crude material. 5.MAbs have the ability to behave in its normal physiological way to attack foreign material, thus finding place in defense against infection. Monoclonal antibody based products are covered by a number of recent compendia. The in vivo application of MAbs is based on critical factors listed below in the table. Antibody related Affinity Specificity Class and subclass Murin vs. human Intact vs. fragments Dose Vascularity Target related Distribution of antigens Antigenic heterogeneity Antigenic modulation Circulating antigen Route of administration Immunogenicity 17 SKPCPER

24 For effective localization, the monoclonal antibodies should have high affinity for the target antigen. Cross-sensitivity with non-target tissue should be minimal. The class and subclass of the immunoglobulin affect biodistribution and blood clearance as well as interaction with immune mechanisms of the host. Blood clearance and catabolism are generally faster with heterogonous murine immunoglobulins as compared to homologous human MAbs. The lower the molecular weight of the antibody, the faster the blood clearance. Intact murin IgG has a blood half-life of approximately 30h.and catebolized in liver and spleen, as Well as other sites in the body. F (ab ) 2 and Fab/Fab' have half lives of approximately 20h.and 2 h.respectivly, and tend to accumulate in kindly. Due to their smaller molecular size, fragments are better able to diffuse out of capillaries and onto target tissue. However, for intension with immune effector function of the host, intact immunoglobulin with the Fc portion is required. Antiboby does also have an effect on blood clearance and biodistribution. Higher does generally at 10mg or above, appears to saturate non-specific sites in the liver and the other organs, as a consequence, more antibodies remain in the blood and clearance is slower. Routes of injection other than intravenous have also been shown to alter biodistribution. Intralymphatic and intraperitoneal administrations tend to concentrate and prolong the dose at these sites and lead to lower systemic levels. Characteristics of target tissue are also important in MAb localization. The tissue should be well vascularized or directly exposed to blood system. This is often a problem with solid tumors, which are poorly vascularized. The antigen in the target tissue should be densely and uniformly expressed and accessible to antibody binding. Viable cells must express target antigen on the cell surface, whereas MAbs that localize dead cells may depend on reactivity with intracellular antigens. Tumor cells often demonstrate antigenic heterogenety. In some causes the complex to be shed or internalized, rendering the cell devoid of expressed antigen. Cross reactive target antigen may also be found in the circulation, often 18 SKPCPER

25 as a consequence of tumor cell shedding. This circulating antigen could complex with the injected MAb and blocks it from binding to the target site. The application of monoclonal antibodies can therefore be classified as: A. Diagnostic uses 1.Diagnostic reagents 2. Diagnostic imaging a) Cardiovascular diseases b) Sites of bacterial infection c) Cell surface markers d) Detection of circulating antigens e) Cancer f) Hormones g) Immunometric assay procedures B. Therapeutic application 1.General a) MAb itself b) Radioisotope immunoconjugates c) Toxin drug conjugates 2.Transplantation a) Organ b) Bone marrow 3.Infectious diseases a) Micro-organisms b) Parasites 4.Enzyme and proteins 5.Cardiovascular diseases 6.Autoimmune diseases 19 SKPCPER

26 7.Cancer 8.Antibodies C. Investigational and analytical application 1.Lymphocyte phenotype 2. Purification of proteins 3. Radio immunoassays D. Drug Targeting 1.Immunotoxins 2.Suppresser deletion 3.Site-specific modification 4.antibody enzyme conjugates E. Miscellaneous 1.abzymes 2.Autoantibody fingerprinting 20 SKPCPER

27 Clinical Applications of Monoclonal Antibodies Hematologic malignancies There are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies. One of the most popular target antigens is CD20, found on B cell malignancies. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia. The CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapy-resistant hairy cell leukemia. Two new monoclonal antibodies targeting CD20, tositumomab and ibritumomab, have been submitted to the Food and Drug Administration (FDA). These antibodies are conjugated with radioisotopes. The first monoclonal antibody to receive FDA approval was rituximab. Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen, a signature B cell antigen. CD20 has an important functional role in B cell activation, proliferation, and differentiation. This antigen is a transmembrane protein composed of 297 amino acids. The intracellular portion contains phosphorylation sequences for protein kinase C, calmodulin, and casein kinase 2 CD20 is thought to act as a calcium channel as well, given the great structural homology between the CD20 protein and the calcium channels. When CD20 was introduced into cell lines by transfection, an increase in intracellular calcium was observed within the transfected cells. With monoclonal antibody stimulation, we see calcium influx within the cells. Calcium chelators blocked apoptosis induced by CD20 stimulation by monoclonal antibodies. When monoclonal antibodies attach and particularly cross-link CD20 antigen, an increase in intracellular calcium is again observed. This increase appears to activate the SER family of tyrosine kinases, resulting in further phosphorylation of the CD20 inner cytoplasmic chain and also phospholipase C-gamma. At the same time there is an up regulation of C-myc and myb messenger ribonucleic 21 SKPCPER

28 acid (RNA), an increase in adhesion molecule expression and an up regulation of MHC class II proteins. The ultimate result is caspase 3 activation, causing cell apoptosis. As previously mentioned, CD20 is a natural focus for monoclonal antibody therapy because of its relatively high degree of expression in B cell malignancies, perhaps as high as 95% in follicular lymphomas, even with the heterogeneity discussed earlier. The monoclonal antibody rituximab was designed specifically to target CD20. Rituximab is predominantly human (95%). The variable light and heavy chain portion of rituximab is murine, but the remainder is humanized so the formation of human anti-mouse antibody is not significant. Rituximab is thought to induce cell apoptosis by inducing calcium influx, releasing caspase activity. In addition, evidence of indirect effects through ADCC and CDC has been observed. Rituximab is indicated for treatment of low-grade lymphomas refractory to conventional chemotherapy. Based upon this work it has been evaluated for firstline and combination therapy. Results of studies using rituximab as first-line treatment of low-grade non-hodgkin lymphoma have been encouraging Patients who had not received any prior therapy were treated with rituximab 375 mg/m 2 on a weekly basis for 4 weeks and then re-evaluated 2 weeks posttherapy. The patients who achieved a complete or partial response, or who had stable disease received rituximab maintenance therapy (weekly for 4 weeks every 6 months). Patients who showed evidence of progression were taken off maintenance therapy. At the time of initial re-evaluation at 6 weeks, 54% of the patients showed objective response to treatment. An additional 36% had stable disease or minor response. At the time of publication 13 patients had undergone a second course of treatment, and 4 additional responses were documented. Four patients improved from partial to complete response. Treatment with rituximab was well 22 SKPCPER

29 tolerated, with only 1 of the 39 patients experiencing grade 3-4 infusion related toxicity. These responses were durable as well. For patients who achieved partial or complete response, one-year follow-up showed no evidence of disease progression. One-year survival was 69%; survival at two years 67%. While overall survival is not an unusual finding in low-grade lymphoma, the duration of response remains relatively impressive. Rituximab has been combined with conventional chemotherapy for patients with intermediate grade or diffuse large cell non-hodgkin lymphoma. CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) is standard therapy for this type and stage of disease. In a multi-institutional study, 33 patients with newly diagnosed large cell lymphoma received six infusions of rituximab 375 mg/m 2 on day 1 of each cycle combined with six doses of CHOP on day 3 of each cycle. The overall response rate was 94%, with 20 patients (61%) achieving a complete response. Eleven patients (33%) experienced partial response, and 2 patients were found to have progressive disease. Median duration of response and time to progression had not been reached after a median follow-up time of 26 months. Twenty-nine patients remained in remission during this observation period. The most frequent adverse events associated with rituximab were fever and chills, primarily during the first infusion. The investigators concluded that rituximab did not appear to increase the toxicity of therapy. These results are interesting for two reasons. First, the number of partial responses to combination therapy was greater than with rituximab alone. Second, the responses even the partial ones tended to be durable. 23 SKPCPER

30 Solid tumors Compared to hematologic malignancies, solid tumors do not have as many specific targets for monoclonal antibodies that are not cross-reactive with antigens on normal tissues. Two significant monoclonal antibodies have been used in solid tumors: edrecolomab and trastuzumab. Edrecolomab targets the 17-1A antigen seen in colon and rectal cancer, and has been AP jority had either lung or liver metastasis. All patients received a loading dose of trastuzumab (4 mg/kg), followed by weekly maintenance therapy of 2 mg/kg until evidence of disease progression. Primary endpoints were tumor assessment relative to response. Secondary endpoints were duration of response, time to tumor progression, time to treatment failure, and quality of life. As first-line monotherapy, trastuzumab has demonstrated efficacy and safety in patients with metastasis breast cancer. This study included 114 women with HER-2-overexpressing metastasis breast cancer with no prior chemotherapy. Patients were randomized to receive a loading dose of trastuzumab 4 mg/kg followed by 2 mg/kg weekly, or an 8 mg/kg loading dose followed by 4 mg/kg weekly. Primary endpoint was overall response rate. Secondary endpoints were disease relapse, time to tumor progression, and overall survival. These investigators found no benefit to the higher versus lower dose of trastuzumab. However, they noted a difference in response that correlated to HER-2 over expression. Over expression was measured by immunohistochemistry (IHC) and then rechecked with fluorescent in situ hybridization (FISH). Interestingly, patients who were positive for over expression by IHC were not necessarily positive by FISH. Over expression as confirmed by FISH was strongly correlated to response, and these patients appeared to have garnered the most clinical benefit from treatment with trastuzumab. 24 SKPCPER

31 Monoclonal Antibodies Used to Treat Cancer MAb Name Trade Name Used to Treat: Rituximab Rituxan Non-Hodgkin lymphoma 1997 Trastuzumab Herceptin Breast cancer 1998 Gemtuzumab ozogamicin* Alemtuzumab Mylotarg Campath Acute myelogenous leukemia (AML) Chronic lymphocytic leukemia (CLL) Approved in: Ibritumomab tiuxetan* Zevalin Non-Hodgkin lymphoma 2002 Tositumomab* Bexxar Non-Hodgkin lymphoma 2003 Cetuximab Bevacizumab Erbitux Avastin Colorectal cancer Head & neck cancers Colorectal cancer Non-small cell lung cancer Panitumumab Vectibix Colorectal cancer SKPCPER

32 Therapeutic Monoclonal Antibodies GENERIC NAME TRADE NAME COMPANY INDICATIONS DOSE before surgery and 12 mg/m 2 4 days later. ROUTE Basiliximab Simulect Novartis Prevention of Adult: 20 mg IV renal within 2 hours infusion transplant prior to rejection transplantation then 20 mg 4 days after transplantation Child: 12 mg/m 2 Daclizumab Zenapax Roche Prevention of 1 mg/kg before IV renal and 2, 4, 6 and 8 infusion transplant weeks after rejection transplantation. Gemtuzumab Mylotarg Ozogamicin Wyeth- Ayerst Treatment of 9 mg/m 2 repeated IV acute myeloid 14 days later. infusion leukemia Infliximab Remicade Centocor Treatment of Crohn's disease: IV rheumatoid 5 mg/kg Crohn's infusion arthritis and disease, Crohn's fistulizing: 5 disease mg/kg repeated at 2 and 6 weeks. RA: 3 mg/kg at 0, 2 and 6 weeks then every 8 weeks. Rituximab Rituxan IDEC/ Genentech Treatment lymphoma of 375 mg/m 2 IV IV once weekly for 4 infusion doses (days 1, 8, 15 and 22). Trastuzumab Herceptin Genentech Treatment of 4 mg/kg-loading breast cancer dose; 2-mg/kg maintenance dose. 26 SKPCPER

33 Uses of monoclonal antibodies Monoclonal bodies have a variety of academic, medical and commercial uses. It would be impossible to list all of these here. But the following list should indicate how ubiquitous monoclonal antibody technology has become in biotechnology. Antibodies are used in several diagnostic tests to detect small amounts of drugs; toxins or hormones, e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Another diagnostic uses of antibodies is the diagnosis of AIDS by the ELISA test. Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells. A new cancer drug based on monoclonal antibody technology is Ritoxin, approved by the FDA in November Monoclonal antibodies can be used to treat viral diseases, traditionally considered "untreatable". In fact, there is some evidence to suggest that antibodies may lead to a cure for AIDS. Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria gonorrhoeae can be typed using monoclonal antibodies. Researchers use monoclonal antibodies to identify and to trace specific cells or molecules in an organism, e.g. developmental biologists at tyhe University of Oregon use monoclonal antibodies to find out which proteins are responsible for cell differentiation in the respiratory system. OKT3, an antibody to the T3 antigen of T cells, is used to alleviate the problem of organ rejection in patients who have had organ transplants. 27 SKPCPER

34 A Advantages of monoclonal antibodies Monoclonal antibodies are of exceptionally high quality; represents only one molecular species and which may be obtained virtually in a homogeneous state conventional antiserums possess certain disadvantages, for e.g, they consist of a mixture of antibodies and major portion of the sample contain irrelevant immunoglobulin. This properties of conventional antiserum lead to cross reactions with other antigens summary of the advantages of Mabs over conventional antisera is listed below: 1. Pure one molecular species only. 2. Specificity for one antigenic determinant. 3. Cross-reactions means shared determinants. 4. Antiserums titer values obtained are very high. 5. Antibodies with high avidity can be produced. 6. Immure immunogen can be used. 7. In vitro or in vivo production is possible with high production rates. 8. Maintenance of farm/animals oils not required for immunization and bleeding. 9. Immortal cell lines. 10. Antiserum having identical antibody with an identical specificity and constants properties can be obtained worldwide. 11. High reproducibility with respect to specificity and avidity. 12. Production of cell lines to individual components of a mixture. 13. Radiolabelling and fluorescent conjugation or enzyme marking of Mabs is easy. 28 SKPCPER

35 Limitation of monoclonal antibodies The first and formost limitation is the initial cost involved in the technique. However, on continues production, the cost is less as compared to conventional antiserum production. The method is time consuming and has its own drawbacks. Precipitate formation In general, Abs donot form a precipate in a standard double-immunodiffusion method. This leads to the necessity to produce a lattice framework for precipitation in tests like ouchterlony assays. Complement fixation Conventional antiserum possess better compliment fixing capabilities than do MAbs.For an efficient compliment fixation, binding of C1q component to two antibody, the molecules (Fc region) are required either adjacent to them or nearby to determinants. Abs produced via cell fusion contains mainly IgG class. Yet, they show poor complement fixing capabilities. The reason given os related the population density of antigenic determinants present. Henceforth, a blend of MAbs required producing the necessary synergism. Antibody specificity Production of MAbs is against a single antigenic determinant principally incorporates high level of selective specificity in to the MAbs thus rendering them incapable to distinguish between a groups of different molecules cells bearing the 29 SKPCPER

36 chemical structure or determinats except one against which they are raised. A conventional antiserum contains antibodies to all the determinants on an antigen and can be precisely used as 'fingerprints' identification for that antigen. However, complete cross-reaction can occur for MAbs and may pose problems in assays where one molecular species amongst several very similar molecular entities is to be detected. The well-identified problem of cross reactivity could effectively be addressed by production of determinants specific antibody clone. The approached adds to the precision and efficiency of MAbs analytical techniques. Antibody avidity The energy of binding to an antigen is 'precise' in case of MAb whereas it is 'average' in case conventional an antiserum. The high antibody avidity of a MAb has advantages as well as disadvantages. It is advantageous in case of immiunoassay methods and undesirable for purification process (affinity chromatography). 30 SKPCPER

37 References 1) < mes.html>: Accessed Feb. P/S/L Consulting Group, Inc Combination HIV-IG/monoclonal antibody dose neutralizes AIDS virus. 2) < htm>: Accessed Feb. Orrs, A FDA approves monoclonal antibody for cancer treatment. 3) < Accessed Feb. Transweb Monoclonal antibodies and OKT3. 4) < Accessed Feb. Wang SP, Holmes KK, Knapp JS, Ott S, Kyzer. Immunologic classification of Neisseria gonorrhoeae with immunofluorescence. J Immunol 1977; 119: ) Anon. Mylotarg (Gemtuzumab Ozogamicin) Approved By FDA for Leukemia. Doctor's Guide May 18, 2000 ( dg/1d27c6.htm). 6) Anon: Acute myeloid leukaemia-new drug gets a bull's-eye view. Inpharma 2000; 1223:7. 7) Anon: Gemtuzumab for relapsed acute myeloid leukemia. Medical Letter on Drugs & Therapeutics 2000; 42(1083): ) Basiliximab, Drugdex Drug Evaluations, Micromedex Intranet Healthcare Series, September, ) Biotech Resources Monoclonal antibody technology. < Accessed Feb. 31 SKPCPER

38 10) Biotech Resources Monoclonal antibody technology -- the basics. < Feb. Accessed 11) Biotech Resources Life ScienceDictionary. < Feb. Accessed 12) Breedyeld FC. Therapeutic monoclonal antibodies. Lancet (9205): ) Campbell, N.A Biology - 4th Ed. The Benjamin/Cummings Publishing Co., CA, pp ) Chaudhuri, T.R., Zinn, K.R., Morris, J.S., McDonald, G.A., Llorens, A.S., and Chaudhuri, T.K Human monoclonal antibody developed against ovarian cancer cell surface antigen. 15) Daclizumab, Drugdex Drug Evaluations, Micromedex Intranet Healthcare Series, March, ) Emerging clinical applications of monoclonal antibodies in cancer Immunotherapy. Biotechnology Online. Fox, DA. Cytokine blockade as a new strategy to treat rheumatoid arthritis: Inhibition of tumor necrosis factor. Arch Int Med 2000; 160: ) Hakimi J, Mould D, Waldmann TA, et al. Development of Zenapax: A Humanized Anti-Tac Antibody, in Antibody Therapeutics, Harris WJ, Adair JR (eds), CRC Press, Boca Raton, 1997, Chapter ) y/cad/abstract8127.html: Accessed Feb. Fratella, J Research Probes Custom Made in Monoclonal Antibody Facility. 20) Knight DM, et al: Construction and initial characterization of a mousehuman chimeric anti-tnf antibody. Molec Immunol 1993; 30: ) Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: PMID Reproduced in J Immunol 2005; 174: PMID SKPCPER

39 22) Mylotarg (Gemtuzumab Ozogamicin), Wyeth-Ayerst Pharmaceuticals, Philadelphia, PA, June, ) Novartis product page for Simulect (basiliximab for injection). Retrieved ) ORTHOCLONE OKT 3 Information page at Ortho Biotech. Retrieved ) Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature 1988;332:323 26) S.P.VYAS, V.K.DIXIT, Pharmaceutical biotechnology,monoclonal antibodies & hybridoma technology. January 1998, page no: ) Sievers EL, Appelbaum FR, Spielberger RT, et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: A phase I study of an anti-cd33 calicheamicin immunoconjugate. Blood 1999; 93: ) Simulect (basiliximab) Product Monograph, Novartis, ) Van Hogezand RA, Verspaget HW: The future role of anti-tumor necrosis factor-" products in the treatment of Crohn's disease. Drugs 1998; 56: ) Waldmann, Thomas A. (2003). Immunotherapy: past, present and future. Nature Medicine 9, ) ZENAPAX (daclizumab) Information page at Roche Pharmaceuticals. Retrieved SKPCPER