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1. Monoclonal Antibodies ( mAbs ): LECTURE 6

2. History Of Discovery Discovery of tetanus and diphtheria antitoxins 2 Paul Ehrlich proposes “Side-Chain Theory” for antibody & antigen (lock & key) interaction 3 Linus Pauling confirms lock & key theory 4 Astrid Fagraeus discovered that B cells (plasma cells) were responsible for generating antibodies 5 César Milstein and Georges Köhler develop method of producing "custom” antibodies in vitro, by producing a hybridoma 1 First mAb approved for clinical use in transplant rejection: Muromonab-CD3 – a mouse antibody 1,2 Abciximab – first chimeric antibody (fragment) 2 Daclizumab – first humanised mAb (transplant rejection) 2 Adalimumab - 1 st fully human mAb approved by FDA 2 1897 1890 1940s 1901 Nobel Prize Emil Adolf von Behring 6 1908 Nobel Prize Paul Ehrlich, Ilya Mechnikov 6 1954 Nobel Prize Linus Pauling 7 1984 Nobel Prize César Milstein, Niels Jerne, Georges Köhler 6 1948 1975 1986 1994 1997 2002 1. Catapano AL, et al. (2013). Atherosclerosis, 228(1):18-28; 2. Foltz I, et al . Circulation 2013 Jun 4;127(22):2222-30; 3. Prüll C Med Hist . 2003 Jul;47(3):332-56; 4. Gormley M Endeavour . 2007 Jun;31(2):71-7; 5. LeBien TW & Tedder TF Blood . 2008 Sep 1;112(5):1570-80 ; 6. Nobelprize.org (2014) All Nobel Laureates in Physiology or Medicine. Available at: www.nobelprize.org/nobel_prizes/medicine/laureates/ Accessed: July 2014 7. Nobelprize.org (2014). All Nobel Laureates in Chemistry. Available at: www.nobelprize.org/nobel_prizes/chemistry/laureates/ Accessed: July 2014

3. Monoclonal Antibodies Monoclonal antibodies are used in: Serological identification ( diagnostic ) tests To prevent tissue rejections Anti IL-2 (T- and B-cell proliferation) , Basiliximab have been used to treat rheumatoid arthritis in more than a million patients. Which reduce proinflamatory cytokine cascades; help to alleviate pain, stiffness, and joint swelling; and promote healing and tissue repair. Anti TNF-α golimumab To make immunotoxins to treat cancer. Trastuzumab Immunotoxins can be made by combining a monoclonal antibody and a toxin diphtheria toxin ; The antibody localize the target (antigen) The toxin will then kill a specific antigen .( toxin part will destroy the cell) Anti growth factor receptor e.g , anti HER-2 for breast cancer Trastuzumab

4. Nomenclature of Therapeutic Antibodies Terminate the name in – ximab for chimeric antibodies and – umab for humanized antibodies.

5. Antibodies Antibodies are naturally occurring proteins that help protect against infectious disease Constant region Fc Fab Heavy chains Light chain Light chain Variable region Fab Sompayrac L (2012). How The Immune System Works. Hoboken: Wiley-Blackwell.

6. B-cell antibody receptors recognise antigens Antigens are molecules that cause an organism to generate antibodies B-cells activate when their antibody receptors bind antigen Activated B-cells differentiate into plasma cells and memory B-cells Plasma cells secrete antibodies B-Cells Produce Antibodies Y Y Y B-cell Activated B-cell Plasma cell Memory B-cell Secreted antibodies Y Y Y Y Y Y Antigen Y Y Y Y Y Y Y Y Antibody receptors Sompayrac L (2012). How The Immune System Works. Hoboken: Wiley-Blackwell.

7. Immunogenicity Fully Mouse 1 st generation Chimeric 2 nd generation Humanised 3 rd generation “Fully” Human 4 th generation Highly immunogenic 100% Mouse Still immunogenic ~30% Mouse e.g. ritu ximab and abci ximab Still immunogenic ~5-10% Mouse e.g. trastu zumab and bevaci zumab Least immunogenic e.g. adalim umab and panitum umab e.g. ibritum omab Mouse variable Mouse constant Human variable Human constant 1. Foltz I et al . Circulation 2013 Jun 4;127(22):2222-30; 2. Nelson AL et al . Nature Reviews Drug Discovery 2010 Oct;9(10):767-74. Monoclonal Antibody Evolution

8. Large Molecule (Biologic) 1 Small Molecule (Drug) 1 Extremely high specificity 2 Good specificity 2 Parenteral administration 3 Commonly administered orally 3 Eliminated primarily by cellular endocytosis, phagocytosis and target-mediated clearance 3,4 Metabolised and eliminated primarily by liver and kidneys 3,4 Unlikely to have drug-drug interaction 4 May have drug-drug interactions 4 Longer half-life, less frequent administration 4 Shorter half-life, more frequent administration 4 Produced by genetically engineered cells or purified from natural sources 3 Synthesised chemically or purified from natural sources 3 Typically do not cross blood-brain barrier 5 Some cross blood-brain barrier 5 Can be immunogenic 4 Rarely immunogenic 4 Biologic And Small Molecule Drugs 1. Generics and Biosimilars Initiative. (2012, June 29). Small molecule versus biological drugs . Accessed http://www.gabionline.net/Biosimilars/Research/Small-molecule-versus-biological-drugs (17 th July 2014) ; 2. Webb, D.R., et al. (2013). Biochemical Pharmacology , 85(2):147-152; 3. Vugumeyster Y et al. (2012). World Journal of Biological Chemistry, 3 (4), 73-92; 4. Catapano , AL et al. (2013). Atherosclerosis, 228(1):18-28; 5. Gabathuler (2010). Neurobiology of Disease , 48-57.

9. Multiple B-Cells Generate Antibodies That Bind Different Regions Of The Antigen Y Y Y Y Y 1. Khanna R (2011) Immunology. Oxford: Oxford University Press; 2. Sompayrac L (2012) How The Immune System Works. Hoboken: Wiley-Blackwell. Plasma B-cells producing antibodies

10. Epitope 1 Epitope 2 Epitope 5 Epitope 3 Epitope 4 Multiple B-Cells Generate Antibodies That Bind Different Regions Of The Antigen Plasma B-cells producing antibodies 1. Khanna R (2011) Immunology. Oxford: Oxford University Press; 2. Sompayrac L (2012) How The Immune System Works. Hoboken: Wiley-Blackwell.

11. Epitope 1 Epitope 2 Epitope 5 Epitope 3 Epitope 4 Polyclonal Vs. Monoclonal Antibodies Y Y Y Y Polyclonal antibody Monoclonal antibody Y Y Y Y 1. Khanna R (2011) Immunology. Oxford: Oxford University Press; 2. Köhler G, C Milstein (1975)  Nature   256 :495-497. Y Y

12. Monoclonal Antibodies In The Clinic Monoclonal antibodies were first introduced into clinical practice in 1986 2 Over 30 monoclonal antibodies are approved for clinical use by European and US regulatory agencies for a wide variety of indications, including but not limited to 2 : Asthma Autoimmune diseases Oncology Ophthalmic disorders Approximately 235 monoclonal antibodies are in active PIII trials for a wide variety of indications, including but not limited to 3 : Alzheimer’s disease Autoimmune diseases Cardiovascular disease Infectious disease Osteoporosis Cumulative number of human monoclonal antibodies entering clinical study between 1985 and 2008 1 100 1985 1993 2001 2007 Number of clinical candidates Year 90 80 70 60 50 40 30 20 10 110 120 130 140 150 1989 1997 2005 2003 1999 1995 1991 1987 All human monoclonal antibodies Antineoplastic only Immunomodulatory only Anti-infective only Other indications 1. Adapted from: Nelson AL et al. Nat Rev Drug Discov 2010;9:325–38; 2. Landes Bioscience (2014). mAbs : About this journal. Available at: http://www.landesbioscience.com/journals/mabs/about/. Accessed July 2014; 3. ClinicalTrials.gov (July 2014). Available at: http://www.clinicaltrials.gov/.

13. Cardiovascular Monoclonal Antibodies EMA Approved 1 mAb Name Year Indication Comment Trade Name Muronomab-CD3 1986 Acute heart transplant rejection 1 st mAb approved; murine monoclonal antibody targeting CD3 Orthoclone OKT3 Abciximab 1995 GpIIb-IIIa Antiplatelet Chimeric Fab ReoPro 1. Landes Bioscience (2014). mAbs : About this journal. Available at: http://www.landesbioscience.com/journals/mabs/about/. Accessed July 2014; 2. MHRA (2014). DigiFab . Available at: http://www.mhra.gov.uk/home/groups/par/documents/websiteresources/con126289.pdf. Accessed July 2014

14. Peptides 2. Immunization Neg 2nd immunization 1st immunization 14 days Serum testing 14 days 10 days 3rd immunization Proteins cDNA Cell lines Different types of antigen 4th immunization FUSION 3 days Pos

15. Validated targets Disease pathology Receptor Signalling molecule Cell Nucleus Receptor Signalling molecule 1. Foltz, I., et al . (2013). Circulation , 127:2222-2230; 2. Hughes, J. (2011). Principles of early drug discovery. British Journal of Pharmacology , 1239-1249. Target Discovery

16. B-cells Myeloma cells Hybridoma cells Spleen Single epitope Bind the same epitope From a single B-cell; have genetically identical variable regions Multiple selection options Hybridoma Phage display Genetically engineered mice Monoclonal antibodies Receptor 1. Köhler G, C Milstein.(1975).  Nature   256 :495-497. Monoclonal Antibodies

17. hypoxanthine-aminopterin-thymidine" HAT medium Hypoxanthine: This is a precursor to adenine, a crucial component of DNA and RNA. Cells can utilize hypoxanthine as a source of adenine if they lack the ability to synthesize it themselves. Aminopterin: Aminopterin is a compound that inhibits the synthesis of nucleic acids (DNA and RNA) by blocking the action of an enzyme called dihydrofolate reductase. This inhibition disrupts the ability of cells to replicate their DNA and divide. Thymidine: Thymidine is a nucleoside that is essential for DNA synthesis. It can bypass the blockage caused by aminopterin, allowing cells to continue replicating DNA despite the inhibition of nucleic acid synthesis. In HAT medium : Myeloma cells cannot grow because they lack HGPRT and cannot salvage hypoxanthine to make adenine. Antibody-producing B cells (spleen cells or lymphocytes) can survive because they have HGPRT and can utilize hypoxanthine. Hybridoma cells, resulting from the fusion of myeloma cells and antibody-producing cells, inherit the ability to produce antibodies from the antibody-producing cells and the ability to survive in HAT medium from the myeloma cells. This allows only the hybridomas to grow and survive in the HAT medium

18. 3. Fusion HAT Selection + HPRT- HPRT- HPRT- HPRT- HPRT-

19. 3 . Hybridomas selection (I) More than 24.000 hybridomas 10 days in the incubator 1 day 3 days 5 days 7 days 9 days 10 days S C R E E N I N G

20. 3 . Hybridomas selection (II) ELISA Immunohistochemistry WB/IP Flow Cytometry

21. Genetically engineered mouse with human antibody gene Human antibody gene added Scientists use genetic engineering to create mice to contain a human antibody gene When injected with antigen, genetically engineered mice produce fully human antibodies Fully human antibody Mouse antibody gene silenced Mouse embryo X Receptor 1. Frenzel A et al . (2013). Expression of recombinant antibodies. Frontiers in Immunology , 4, 217. Humanising Mouse Antibodies: Genetically Engineered Mice

22. Y Y Y B-cell Human variable Human constant Y Y Y Selected phage Human variable Human constant 1. Rodrigues ME et al . (2013). J Microbiol Biotechnology , 23(9):1308-21 Selected Monoclonal Antibody Gene Transferred To Chinese Hamster Ovary (CHO) Cells For Manufacture

23. Insertion of antibody gene into CHO cell for manufacture Selected Monoclonal Antibody Gene Transferred To Chinese Hamster Ovary (CHO) Cells For Manufacture Y Y Y B-cell Y Y Y Selected phage 1. Rodrigues ME et al . (2013). J Microbiol Biotechnology , 23(9):1308-21

24. CHO: Chinese Hamster Ovary Cells Cell line established in 1957 by Dr. Puck at the University of Colorado Most widely-used mammalian commercial production line HIV, influenza , polio, herpes, and measles do not replicate in CHO CHO cells 1. Jayapal KP et al . (2007). Recombinant Protein Therapeutics From CHO Cells — 20 years and Counting. Chem. Eng. Prog .,   103  (10): 40–47

25. Therapeutic Monoclonal Antibody Manufacturing Upstream Process Industrial scale operation CHO cells produce monoclonal antibodies Small scale culture Small scale bioreactor Larger scale bioreactor Stock culture ~100 mL ~1 L ~50 L ~3,000 L ~10,000 L 1. Daugherty E (2012) Biotechnology St. Paul: Paradigm Publishing, Inc.

26. Therapeutic packaged Column chromatography Centrifuge Filtration Formulation, filtration & fill Therapeutic Monoclonal Antibody Manufacturing Downstream Process 1. Daugherty E (2012) Biotechnology St. Paul: Paradigm Publishing, Inc.

27. Conclusion Monoclonal antibodies are used for a wide variety of clinical indications Therapeutic monoclonal antibodies can be selected by use of hybridoma technology, phage display or genetically engineered mice Humanised and fully human monoclonal antibodies have lower incidence of immunogenicity than mouse monoclonal antibodies Once the monoclonal antibody is selected, the monoclonal antibody gene is transferred to mammalian cells for manufacture Monoclonal antibodies can tag a cell for destruction, block cell receptors to interrupt disease pathology or capture signaling molecules to interrupt disease pathology