MONOCLONAL ANTIBODY.pptx

monoclonal Antibidy ppt

1. Presented By - MOUSAM BHOWMIK M. PHARM, 1 ST YEAR 2 ND SEM ACHARYA & BM REDDY COLLEGE OF PHARMACY MONOCLONAL ANTIBODIES Presented To Dr. Joysa Ruby J Assistant professor

2. What is Antibody? Antibodies are glycoprotein molecule present in the serum. They are produced in response to antigen which are either protein or polysaccharide molecules which may be foreign to the body. Antibodies are secreted by a class of blood cells known as B-lymphocytes. Each antibody produced is specific to that particular antigen which has stimulated its production.

3. Antibodies are also called as Immunoglobulin (Ig) and the structure of antibody contains two chain such as – Heavy chain, Light chain and constant region ,variable region and they also contain di sulphide bond etc.

4. What is Monoclonal Antibodies? Monoclonal antibodies (mAbs) are defined as “laboratory-produced molecules engineered to serve as substitute antibodies that can restore, enhance, or mimic the immune system's attack on cancer cells” by binding to antigens found on the surface of cancer cells. In the 18th century, it was discovered by Dr Edward Jenner that “fluid obtained from a smallpox pustule when injected into a recipient provided immunity from acquiring the disease.

5. Structure of monoclonal antibody

6. Mechanism of action The understanding of how these antibodies target cancer cells has helped to revolutionize the methods used to treat cancer and resulted in a more tolerable toxicity profile than standard chemotherapy. When utilizing mAbs in oncology, several mechanisms of action exist to destroy the cancer cells. These mechanisms include impeding tumor cell survival cascades, inhibiting tumor growth by interfering with tumor angiogenesis, eluding programmed cell death, and evading immune checkpoints. Some of the different mechanisms of action include the following:

7. Direct tumor cell killing This action is stimulated by receptor agonist activity, such as an antibody binding to a tumor cell surface receptor and activating it, leading to cell death. It can also be stimulated by receptor antagonist activity “such as an antibody binding to a cell surface receptor and blocking dimerization, kinase activation, and downstream signaling, leading to reduced proliferation and apoptosis.” A mAb binding to an enzyme can lead to neutralization and apoptosis, while conjugated antibodies can be used to deliver a payload, such as a drug, to the tumor cell.

8. 2. Immune-mediated tumor cell killing In this setting, the immune system will seek out cancer cells and destroy them. This type of cell death may be carried out by the initiation of one of several mechanisms. These mechanism. Phagocytosis; complement activation; antibody-dependent cellular cytotoxicity (ADCC); genetically modified T cells being targeted to the tumor by single-chain variable fragment; T cells being activated by antibody-mediated cross-presentation of antigen to dendritic cells; or inhibition of T-cell inhibitory receptors.

9. 3. Vascular and stromal cell ablation This action is also initiated by one of many options. These include: Vasculature receptor antagonism or ligand trapping; Stromal cell inhibition; Delivery of a toxin to stromal cells; or Delivery of a toxin to the vasculature.

10. Classification and Types of mAbs There are four classifications of mAbs: Murine Chimeric humanized human

11. Murine The first mAb to be discovered and reproduced was the murine monoclonal antibody. This type of mAb arises from harvesting B lymphocytes from the spleen of a mice and then fused with an immortal myeloma cell line lacking the hypoxanthine-guanine-phosphoribosyl transferase (HPTR) gene. All of these mAbs are identified with a name that ends in - omab ( ie , muromonab-CD3, blinatumomab, capromab ). Allergic reactions are common when used in humans and often result in the induction of anti-drug antibodies. Murine mAbs also have a short half-life when used in humans because of a relatively weak binding to the human FcR . For oncology, these mAbs may not be the most beneficial as they are “relatively poor recruiters of effector function, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity,” essential functions needed for tumor destruction.

12. b. Chimeric Chimeric mAbs utilize the murine antigen-specific variable region, but the remaining heavy and light chains are human. This was accomplished using genetic engineering techniques, which resulted in mAbs that are approximately 65% human and 35% murine. The chimeric mAbs are identified with names ending in - ximab ( ie , rituximab, infliximab, cetuximab). Compared with their murine counterparts, these mAbs “exhibit an extended half-life in man and show reduced immunogenicity, but nevertheless, the propensity of chimeric mAbs to induce anti-drug antibodies is still considerable”.

13. c. Humanized Humanized mAbs are created by grating the murine hypervariable regions of the light and heavy chains onto a human Ab framework. This results in molecules that are approximately 95% human. This resulted in decreased production of anti-drug antibodies. However, the process to create these molecules is hard and has limitations. These mAbs are identified with names ending in - zumab ( ie , trastuzumab, alemtuzumab, bevacizumab) .

14. d. Human With the development of new technology, fully human mAbs were able to be created. These are created utilizing animals carrying human Ig genes. These transgenes include parts of the variable regions, which enable the recombination of the human antibodies. The animal’s own endogenous Ig genes have been inactivated, enabling the generation of fully human mAbs. These mAbs are less antigenic and better tolerated compared with the other classes of mAbs. Additionally, they appear to remain present in the human body’s circulation compared with the other classes. These mAbs are identified with names ending in - umab ( ie , ofatumumab, daratumumab, denosumab).

15. There are three types of mAbs that are dependent upon how they are administered or used: unconjugated or naked conjugated bispecific

16. Unconjugated mAbs Unconjugated mAbs or “naked” mAbs are those antibodies that function by themselves. These are the most commonly used in the treatment of cancer. In most cases, these molecules attach to antigens on cancer cells. This can illicit one of several responses, which can be that the person’s natural immune response against the cancer cells is enhanced. The mAb attracts immune cells and aids in enhancing the recognition of cancer cells by the immune system, leading to increased apoptosis. Another mechanism is the targeting of immune system checkpoints, while other unconjugated mAbs block antigens on cancers that help them to expand and proliferate

17. 2. Conjugated mAbs When an mAb is combined with a chemotherapy agent or with a radioactive particle it is referred to as a conjugated monoclonal antibody. The mAb serves as a delivery mechanism for the chemotherapy or radioactive particle, circulating through the patient’s body until it finds the intended target antigen. This method helps to minimize harm to normal cells by the chemotherapy agents or radioactive particle attached to the mAb.

18. 3. Bispecific mAbs This unique type of mAb is a combination of two different mAbs which allows for the mAb to attach to two different antigens at the same time. One target is the protein found on cancer cells, and the other target is a protein found on immune cells. This combination allows for the immune cell and cancer cell to be brought together, in hopes of inciting an increased immune response and the destruction of cancer cells.

19. Production of monoclonal antibody Basic steps involved in the production of a monoclonal antibody Immunization (Immunize the animal) Cell fusion process Selection of hybridomas Screening the products Cloning and propagation Characterization and storage

20. Immunize the animal The first step in hybridoma technology is to immunize an animal (mouse), with appropriate antigen. The antigen, along with an adjuvant like Freund’s complete or incomplete adjuvant is injected subcutaneously. Three days prior to killing of the animal, a final dose of antigen is intravenously administered. The concentration of the desired antibodies is assayed in the serum of the animal at frequent intervals during the course of immunization, when optimal the animal is sacrificed.

21. 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.

22. 2. Cell Fusion process The thoroughly washed lymphocytes (spleen cell) are mixed with HGPRT negative myeloma cells. The mixture of cells is exposed to polyethylene glycol (PEG) 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. These cells are composed of a mixture of hybridomas (fused cells), free myeloma cells and free lymphocytes.

23. 3. Selection of Hybridomas When the cells are cultured in HAT medium only the hybridoma cells grow, while the rest will slowly disappear. Selection of a single antibody producing hybrid cells is very important. This is possible if the hybridomas are isolated and grown individually 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.

24. 4. Screening the Products 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. In both the assays (ELISA, RIA) 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

25. 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.

26. 5. Cloning and Propagation The single hybrid cells producing the desired antibody are isolated and cloned. Two techniques are commonly employed for cloning hybrid cells: limiting dilution method soft agar method

27. 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.

28. 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 .

29. Characterization and Storage The monoclonal antibody has to be subjected to biochemical and biophysical characterization. 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.

30. Applications Therapeutic Applications:- Monoclonal antibodies have a wide range of therapeutic applications. MAbs are used in the treatment of cancer, transplantation of bone marrow and organs, autoimmune diseases, cardiovascular diseases and infectious diseases. The therapeutic applications of MAbs are broadly grouped into 2 types: Direct use of MAbs as therapeutic agents MAbs as targeting agents

31. Direct use of MAbs as therapeutic agents Monoclonal antibodies can be directly used for enhancing the immune function of the host. Direct use of MAbs causes minimal toxicity to the target tissues or the host. In destroying disease-causing organisms: MAbs promote efficient opsonization of pathogenic organisms (by coating with antibody) and enhance phagocytosis. In fact, MAbs were found to protect chimpanzees against certain viral (hepatitis B-virus) and bacterial (E. coli Haemophilus influenza, Streptococcus sp and Pseudomonas sp ) infections.

32. In the treatment of cancer : MAbs , against the antigens on the surface of cancer cells, are useful for the treatment of cancer. The antibodies bind to the cancer cells and destroy them. This is brought out by antibody—dependent cell-mediated cytotoxicity, complement-mediated cytotoxicity and phagocytosis of cancer cells (coated with MAbs ) by reticuloendothelial system.

33. The patients suffering from leukemia, colorectal cancer, lymphoma and melanoma have been treated with MAbs . However, there was a wide variation in the success rate. A monoclonal antibody specific to the cells of leukemia is used to destroy the residual leukemia cells without affecting other cells. MAbs are used in vitro to remove the residual tumor cells prior to autologous bone marrow transplantation (transplantation of the patient’s own bone marrow cells, due to non-availability of a suitable donor).

34. Limitations for direct use of MAbs in cancer: The MAbs produced in mice and directly used for therapeutic purposes may lead to the development of antimouse antibodies and hypersensitivity reactions All the cancer cells may not carry the same antigen for which MAb has been produced. Thus, MAbs may not be attached to some cancer cells at all. The free antigens (of target cells) present in the circulation may bind to MAbs and prevent them from their action on the target cells

35. In the treatment of AIDS : Immunosuppression is the hall mark of AIDS. This is caused by reduction in CD4 (cluster determinant antigen 4) cells of T-lymphocytes. The human immunodeficiency virus (HIV) binds to specific receptors on CD4 cells by using surface membrane glycoprotein (gp120). Genetic engineers have been successful to attach Fc portion of mouse monoclonal antibody to human CD4 molecule. This complex has high affinity to bind to membrane glycoprotein gp120 of virus infected cells.

36. In the treatment of autoimmune diseases : Autoimmune diseases like rheumatoid arthritis and multiple sclerosis are of great concern. Some success has been reported in the clinical trials of rheumatoid arthritis patients by using MAbs directed against T-lymphocytes and B-lymphocytes.

37. MAbs as Targeting Agents in Therapy Toxins, drugs, radioisotopes etc., can be attached or conjugated to the tissue-specific monoclonal antibodies and carried to target tissues for efficient action. This allows higher concentration of drugs to reach the desired site with minimal toxicity. In this way, MAbs are used for the appropriate delivery of drugs or isotopes.

38. MAbs in drug delivery :- In general, the drugs are less effective in vivo (in the living body) when compared to in vitro (in laboratory when tested with cultured cells). This is mainly due to the fact that sufficient quantity of the drug does not reach the target tissue. This problem can be - solved by using tissue-specific MABs. MABs. The drugs can be coupled with MAb (directed against a cell surface antigen of the cells, say a tumor) and specifically targeted to reach the site of action.

39. MAbs in radio immunotherapy (RAIT): The radioisotopes can be coupled to MAbs that are directed against tumor cells. This allows the concentration of radioactivity at the desired sites and a very efficient killing of target cells (tumor cells). The advantage with radio immunotherapy is that conjugated complex need not penetrate the cells, as is required in immunotoxin therapy. The limitation is that the neighboring normal cells may also get damaged or killed. This can be minimized by using radioisotopes with short half-lives. Yttrium-90 with a half-life of 64 hours is a suitable isotope to be employed in RAIT. Due to shortage in the supply of yttrium-90, indium-111 is more commonly used.

40. ADVANTAGE HOMOGENEITY : Monoclonal antibody represents a single antibody molecule that binds to an antigen with the same affinity and promote the same effectors. SPECIFICITY : The product of a single hybridoma reacts with the same epitope on antigen. SELECTION: It is possible to select for specific epitope specificities and generated antibodies against a wider range of antigenic determination. ANTIBODY PRODUCTION : Unlimited quantities of a single well defined monospecific.

41. DISADVANTAGE AFFINITY : average affinity of monoclonal antibodies are generally lower than polyclonal antibodies Effector Function : because antibody is monoclonal, it may produce the desire biologic response. SPECIFICITY : Monoclonal against conformational epitopes on negative proteins may lose reactivity with antigens. CROSS REACTION : Antibodies sometime display unexpected cross reaction with unrelated antigens .

42. FDA approved MABs for Oncology

43. Reference Talreja S, Pandey S, Kumar S. A review on Monoclonal antibody and its application in biotechnology. Journal of Pharmaceutical Sciences and Research. 2020;12(1):49-53. Bayer V. An overview of monoclonal antibodies. InSeminars in oncology nursing 2019 Oct 1 (Vol. 35, No. 5, p. 150927). WB Saunders. Modjtahedi H, Ali S, Essapen S. Therapeutic application of monoclonal antibodies in cancer: advances and challenges. British medical bulletin. 2012 Dec 1;104(1):41-59.