Monoclonal antibody and its delivery

Monoclonal antibody and its delivery helps us treat heart wrecking diseases.

1. Presented by : SHIKHA SINGH Under the guidance of : Dr. Srinivas Mutalik Department of Pharmaceutics Registration No. 160617009 , Manipal College of Pharmaceutical Sciences (MCOPS) MONOCLONAL ANTIBODIES

2. CONTENTS Introduction Structure of monoclonal antiboby Types of monoclonal antibodies Methods for preparation of monoclonal antibodies Applications of monoclonal antibodies Marketed preparations Conclusion References Manipal College of Pharmaceutical Sciences (MCOPS) 2

3. INTRODUCTION Antibodies are glycoprotiens (globulins) present in the serum, also known as immunoglobulins(Ig’s) & are produced by B-lymphocytes Monoclonal antibodies are the monospecific antibodies & are made by identical immune cells that are all clone of a parent cell Manipal College of Pharmaceutical Sciences (MCOPS) 3

4.  Antibodies function as markers, binding to the antigen so that the antigen molecules can be recognized & destroyed by phagocytes Naked mAbs  are antibodies that work by themselves which are non-cytotoxic, drug or radioactive Conjugated mAbs  are linked to a chemotherapeutic drug, radioactive particle, or a cytotoxin , work by take these substances directly to the cells Manipal College of Pharmaceutical Sciences (MCOPS) 4

5. STRUCTURE OF ANTIBODY Y-shaped structure heterodimeric molecules Heavy chain and light chain F ab Fragment and F c Fragment Paratope and Epitope L ight chain Heavy chain FAB region FC region Manipal College of Pharmaceutical Sciences (MCOPS) 5

6. TYPES OF ANTIBODIES Manipal College of Pharmaceutical Sciences (MCOPS) 6

7. Advantages Homogeneity: mAbs represents single antibody molecule that binds to antigens with the same affinity & promote the same effector functions Specificity: The product of a single hybridoma reacts with the same epitope on antigens Selection: It is possible to select for specific epitope specificities Antibody Production: Unlimited quantities of a single well-defined mono-specific antibody can be generated Manipal College of Pharmaceutical Sciences (MCOPS) 7

8. Disadvantages Affinity: Average affinity of monoclonal antibodies is generally lower than polyclonal antibodies. Cross-reactions: Antibodies sometimes display unexpected cross-reactions with unrelated antigens. Time and effort commitment: Very large. Manipal College of Pharmaceutical Sciences (MCOPS) 8

9. PREPARATION METHODS OF mAbs Manipal College of Pharmaceutical Sciences (MCOPS) 9

10. Hybridoma technology Production of mAbs involved: Manipal College of Pharmaceutical Sciences (MCOPS) 10

11. Immunization Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells Manipal College of Pharmaceutical Sciences (MCOPS) ANTIGEN + ADJUVANT SPLEEN REMOVED (source of cells) 11

12. Cell fusion Step 2: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells FUSION HGPRT MYELOMA CELLS SPLEEN CELLS Fused cells, Free myeloma & spleen cells PEG added & washed (3mins) HAT Medium Manipal College of Pharmaceutical Sciences (MCOPS) 12

13. Selection of hybridoma Myeloma cell- HGPRT- & cannot survive in HAT medium B cell (spleen)- HGPRT+ & survive in HAT medium but after some division undergoes normal cell death Hybrid cell (fused)- survive in HAT medium Step 3: - Formation & selection of Hybridoma Cells is based on 2 pathways De-novo pathway & salvage pathway Manipal College of Pharmaceutical Sciences (MCOPS) 13

14. Screening of hybridoma Step 4: - Screening of product ELISA RIA Manipal College of Pharmaceutical Sciences (MCOPS) 14

15. Cloning and propagation of hybridoma Step 5: - Cloning & propagation of product Two techniques are commonly employed for cloning hybrid cells Limiting dilution method or expansion Soft agar method Manipal College of Pharmaceutical Sciences (MCOPS) 15

16. Characterization and storage of hybridoma Step 6: - Characterization & storage of product The monoclonal antibody has to be subjected to biochemical & biophysical characterization for the desired specificity The stability of the cell lines & the MABs are important The cells (and MABs) must be characterized for their ability to withstand freezing & thawing The desired cell lines are frozen in liquid nitrogen at several stages of cloning & culture Manipal College of Pharmaceutical Sciences (MCOPS) 16

17. Manipal College of Pharmaceutical Sciences (MCOPS) 17

18. Large scale production Production of mabs in culture bottles is rather low- 5-10  g/ml Yield can be increased by growing the hybrid cells as ascites in the peritoneal cavity of mice which yields about 5-20 mg of mabs /ml It is superior than the in vitro cultivation techniques Ascitic fluid in mice may have high risk of contamination by pathogenic organism of animal & in addition, several animals have to be sacrificed to produce mabs Hence, many workers prefer in vitro techniques rather than the use of animals Manipal College of Pharmaceutical Sciences (MCOPS) 18

19. Encapsulated hybridoma cells Production of mAb can be substantially increased by increasing the hybridoma cell density in suspension culture By this approach, a much higher concentration of Monoclonal antibody production (10-100  g/ml) can be achieved Manipal College of Pharmaceutical Sciences (MCOPS) 19

20. Alternative methods Using Bacteria as a precursor: Bacteria Antibodies Manipal College of Pharmaceutical Sciences (MCOPS) 20

21. Polymerase chain reaction : Manipal College of Pharmaceutical Sciences (MCOPS) 21

22. APPLICATION Applications of mAbs : mAbs as Target drug delivery Therapeutic uses of mAbs Diagnostic use Manipal College of Pharmaceutical Sciences (MCOPS) 22

23. mAbs as Target drug delivery Dissolution clot Immunoliposomes Immunomicrospheres Radio immunotheraphy Manipal College of Pharmaceutical Sciences (MCOPS) 23

24. Dissolution clot Mechanism mAb is coupled with tPA & used for degradation of blood clots mAb-tPA complex due to a high affinity gets attached to fibrin conversion of plasminogen to plasmin which is in turn dissolves blood clot (fibrin) Clot lysis has been reported by using mAbs-tPA complex in animal Manipal College of Pharmaceutical Sciences (MCOPS) 24

25. Immunoliposomes Manipal College of Pharmaceutical Sciences (MCOPS) 25

26. Immunomicrospheres Biocompatible & biodegradable polymers, antibodies have been conjugated to polymeric microparticles for controlling their in vivo deposition Another study has evaluated the in vivo drug delivery potential of albumin immune-microspheres in mice, microspheres bearing Lewis lung carcinoma MABs demonstrated slightly higher localization in lung carcinoma at 24 h after its administration Manipal College of Pharmaceutical Sciences (MCOPS) 26

27. Radio immunotheraphy Manipal College of Pharmaceutical Sciences (MCOPS) 27

28. Therapeutic uses of mAbs Pregnancy HIV Cancer Autoimmuno diseases Manipal College of Pharmaceutical Sciences (MCOPS) 28

29. Pregnancy A monoclonal antibody can be used to detect pregnancy only 14 days after conception A pregnant woman has the hormone human chorionic gonadotrophin (HCG) in her urine mAb have been produced against HCG to which enzymes is attached, which can later interact with a dye & produce a colour change Manipal College of Pharmaceutical Sciences (MCOPS) 29

30. HIV The test of HIV infection is based on detecting the presence of HIV antibody in the patient’s blood serum Manipal College of Pharmaceutical Sciences (MCOPS) 30

31. Cancer Cancer cells carry specific tumour -associated antigens (TAA) on their plasma membrane. Monoclonal anti-TAA antibodies have been produced Drugs which kill tumour cells or inhibit key proteins in tumour cells are attached to monoclonal anti-TAA antibodies Cancer cells are specifically targeted, avoiding damage to healthy host cells mAbs are being used to track cancer antigens & alone or linked to anticancer agents, to attack cancer metastases Manipal College of Pharmaceutical Sciences (MCOPS) 31

32. Autoimmuno diseases Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease & ulcerative Colitis Basiliximab & daclizumab activated T cells & thereby help preventing acute rejection of kidney transplants Omalizumab inhibits IgE which is useful in moderate-to-severe allergic asthma The monoclonal antibody known as OKT3 is saving organ transplants threatened with rejection & preventing bone marrow transplants from setting off graft-versus-host disease mAbs n eutrilize the action of lymphocytes responsible for the rejection of grafts & destroy the auto-antibodies produced in auto-immune disease. Manipal College of Pharmaceutical Sciences (MCOPS) 32

33. Diagnostic use mAbs can be used to detect for the presence & quantity of this substance, for instance in a Western blot test (to detect a substance in a solution) or an immunofluorescence test mAbs can also be used to purify a substance with techniques called immunoprecipitation & affinity chromatography mAbs allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal & Chlamydia infections Manipal College of Pharmaceutical Sciences (MCOPS) 33

34. Antibody Brand name Type Indication Palivizumab Synagis humanized Respiratory Syncytial Virus Panitumumab Vectibix human Colorectal cancer Ranibizumab Lucentis humanized Macular degeneration Rituximab Rituxan , Mabthera chimeric Non-Hodgkin lymphoma Tocilizumab Actemra Humanised Rheumatoid arthritis Tositumomab Bexxar murine Non-Hodgkin lymphoma Trastuzumab Herceptin humanized Breast cancer

35. Conclusion Monoclonal antibodies are new biological agents that have good clinical effects & an extended choice in the treatment spectrum to the patients who were not responding to the existent treatments New therapeutic approaches are rapidly emerging and further studies may help in designing more specific Mabs that would spare the normal tissue, have less adverse effects and improve the patient’s quality of life Manipal College of Pharmaceutical Sciences (MCOPS) 35

36. References AM Carpbell , monoclonal antibody and immunosensor, edited by P.C. van der VLIET - Department for Physiological Chemistry, University of Utrecht, Utrecht, Volume 23, 1991 Elsevier http://www.biotecharticles.com/Others-Article/Hybridoma-Technology-A-Biotechnology-Technique-378.html,25th Jan 2017 http://pathology.wustl.edu/research/hybridoma.php?page=advantages , 30th Jan 2017 http://en.wikipedia.org/wiki/Monoclonal_antibodies ,27th Jan 2017 Manipal College of Pharmaceutical Sciences (MCOPS) 36

37. Thank you