Antibody engineering by R.S.Priyengha

Engineered Antibodies using New Technology

1. Antibody Engineering BY- Priyengha.R.S M.Sc.,Microbiology , 15KUSM6014 RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 1/22

2. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIOLOG Y 2/22 Introduction Antibodies Structure and its function Methods of Antibody Engineering Hybridoma Method Chimeric Method Humanised Method Applications Conclusion Antibody Engineering CONTENTS

3. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 3/22 Antibody Engineering INTRODUCTION

4. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 4/22 In recent years, antibodies have become increasingly accepted as therapeutics for human diseases, particularly for cancer, viral infection and autoimmune disorders. Monoclonal antibodies ( Mabs ) have been used as diagnostic and analytical reagents since hybridoma technology was invented in 1975. “man-made antibodies.” was named by Cesar Milstein, who was one of the inventors of monoclonal antibody technology. Until the late 1980’s, antibody technology relied primarily on animal immunization and the expression of engineered antibodies. Antibody Engineering Antibody

5. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 5/22 Antibodies Are Made Up Of: 2 Light Chains (identical) ~25 KDa 2 Heavy Chains (identical) ~50 KDa Each Light Chain Bound To Heavy Chain By Disulfide (H-L) Heavy Chain Bound to Heavy Chain (H-H) First 100 a/a Of Amino Terminal Vary of Both H and L Chain Are Variable Referred To As V L , V H , C H And C L CDR ( Complementarity Determining Regions) Are What Bind Ag Remaining Regions Are Very Similar Within Same Class Antibody Engineering Antibody Structure & Functions

6. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIOLOGY 6/22 Repeating Domains of ~110 a/a Intrachain disulfide bonds within each domain Heavy chains 1 V H and either 3 or 4 C H (C H 1, C H 2, C H 3, C H 4) Light chains 1 V L and 1 C L Hinge Region Rich in proline residues (flexible) Hinge found in IgG , IgA and IgD Proline residues are target for proteolytic digestion ( papain and pepsin) Rich in cysteine residues (disulfide bonds) IgM and IgE lack hinge region They instead have extra C H 4 Domain Antibody Engineering

7. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 7/22 Digestion With Papain Yields 3 Fragments 2 identical Fab and 1 Fc Fab - Fragment That is Antigen Binding Fc - Found To Crystallize In Cold Storage Pepsin Digestion F( ab `)2 No Fc Recovery, Digested Entirely Mercaptoethanol Reduction (Eliminates Disulfide Bonds) And Alkylation Showed Antibody Engineering

8. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 8/22 Antibody Engineering

9. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 9/22 Hybridoma development Novel mAb development technology Purification Antibody heterogeneity Recombinant Chimeric antibodies Human antibodies Antibody Engineering Methods of AB Engineering

10. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIOLOGY 10/22 Antibody Engineering Hybridoma Technology

11. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 11/22 Several monoclonal antibody technologies had been developed recently, such as phage display, single B cell culture,single cell amplification from various B cell populations and single plasma cell interrogation technologies. Different from traditional hybridoma technology, the newer technologies use molecular biology techniques to amplify the heavy and light chains of the antibody genes by PCR and produce in either bacterial or mammalian systems with recombinant technology. One of the advantages of the new technologies is applicable to multiple animals, such as rabbit, llama, chicken and other common experimental animals Antibody Engineering Novel Mab Technology

12. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 12/22 The production of recombinant monoclonal antibodies involves repertoire cloning or  phage display/yeast display technologies. Recombinant antibody engineering involves antibody production by the use of viruses or yeast, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected Antibody Engineering RECOMBINANT Technology

13. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 13/22 Antibody Engineering CHIMERIC ANTIBODIES Production of chimeric mouse-human monoclonal antibodies. Chimeric mouse-human heavy- and light-chain expression vectors are produced.These vectors are transfected into Ab myeloma cells. Culture in ampicillin medium selects for transfected myeloma cells that secrete the chimeric antibody. While structurally similar, differences between mouse and human antibodies were sufficient to invoke an immune response when  M urine  monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects and the production of  Human Anti-Mouse A ntibodies  (HAMA ). Recombinant DNA has been explored since the late 1980s to increase residence times. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this " chimeric " or " humanised " DNA through cell culture yielded part-mouse, part-human antibodies.

14. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 14/22 Antibody Engineering CHIMERIC Antibodies Production

15. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 15/22 Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of "fully" human products to reduce the side effects of humanised or chimeric antibodies. Two successful approaches have been identified: transgenic mice and phage display. As of November 2016 , 13/19 cells "fully" human monoclonal antibody therapeutics on the market were derived from transgenic mice technology. These antibodies have been approved to treat cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis and viral infection . Antibody Engineering Humanised Antibodies

16. HUMANIZED ANTIBODIES FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3 CONSTANT CONSTANT CONSTANT FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3 Mouse Human Humanized 16/22 Antibody Engineering RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY

17. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 17/22 Antibody Engineering

18. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 18/22 Antibody Engineering CHIMERIC/HUMANIZED Reduce the HAMA response. Maintain effector functions. Increase the half life of the antibody. Maintain binding affinity. Easy to construct.

19. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 19/22 Antibody Engineering

20. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 20/22 Antibody Engineering

21. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 21/22 Antibody Engineering Applications

22. RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE DEPT OF MICROBIO LOGY 22/22 Antibody Engineering Immunology – Kuby 5 th Edition. Roitt’s Essential immunology by lvan M. Roitt , Peter J. Delves, 10 th edition. Essentials of Medical Pharmacology by KD Tripathi Antibody Engineering Textbook by Carl A.K.Borrebaeck . Antibody Engineering Article By – Hyo Jeong Hong and Sun Taek Kim in Biotechnology and Bioprocess Engineering 2002,7:150- 154 AntibodyEngineering for the development of Therapeutics Antibody –Molecules and cells @ KSMCB 2005 References

23. Thank You….. By Priyengha.R.S II M.Sc., MB.,III sem 15KUSM6014