1. 1 now require 7.6 years on average to complete the clinical development process and receive marketing approval, Total development and approval times for mAbs, one of today's premier biotech tools, compare favorably with small molecule drugs, which require an average of 7.5 years to follow the same path, and with all biotech products, which require an average of 8 years.

2. 2

3. In 2000 antibody revenue was 2-2.4 billion USD Between 2001 and 2002, the market grew by 37% per year, reaching 5.4 billion USD in 2002 It is estimated that 2008 sales will be 16.7 billion USD Antibody revenue over the past few years 3

4. Global Monoclonal Antibodies Market Review 2008 (World Top Ten mAbs) Monoclonal antibodies is one of the current success story of the modern biotechnology R&D. Thirty therapeutic mAbs have been approved around the world, including 23 in the United States. The global sales of monoclonal antibodies were $33 billion in 2008 as compared to $27 billion in 2007. Remicade was the market leader with sales of $6.5 billion in 2008, followed by Rituxan, Herceptin, Avastin and Humira. There were 8 monoclonal antibodies with sales of $1 billion or more in 2008. 4

5. 5 2012 global sales of antibody-based therapeutics exceeded USD50 billion 6 of the top 12 selling drugs in 2012 were antibody-based. The 6 products are: #1 adalimumab ( Humira®), with sales of USD9.265 b #3 rituximab (Rituxan®), with sales of USD 7.285 b; #5 trastuzumab ( Herceptin® ), with sales of USD 6.397 b; #7 infliximab (Remicade®), with sales of USD 6.139 b; #8 bevacizumab (Avastin®), with sales of USD 6.260 b; #11 etanercept (Enbrel®) with sales of USD 4.236 b.

6. Trade nameCompanyTargetSourceYearIndication Orthoclone®Ortho Biotech, Inc. (subsidiary of J&J)CD3all rodent1986Transplantation rejection ReoProTMCentocor, Inc. (subsidiary of Johnson & Johnson) and Eli Lilly GPIIb, IIIachimeric1994High risk angioplasty RituxanTMBiogen Idec and Genentech, Inc.CD20chimeric1994Non-Hodgkin’s lymphoma, rheumatoid arthritis REMICADE®Centocor, Inc. (subsidiary of Johnson & Johnson)TNF-achimeric1998Crohn's disease Simulect®NovartisCD25chimeric1998Transplantation rejection SynagisTMMedimmuneRSV F proteinhumanized1998RSV infection Zenapax®Hoffmann-La Roche Inc., Protein Design LabsCD25humanized1997Transplantation rejection Herceptin®GenentechHER-2humanized1998Breast cancer MylotargTMUCB and WyethCD33humanized2000Acute Myeloid Leukemia Campath®Millenium Pharmaceuticals, Inc. and Berlex Laboratories, Inc. CD52humanized2001Chronic lymphotic leucemia, T-cell lymphoma Zevalin®Idec Pharmaceuticals CorporationCD20murine â with yttrium-90 or indium-111 2002Non-Hodgkin's lymphoma HUMIRATMAbbott Laboratories/Cambridge Antibody TechnologyTNF-ahuman2002Inflammatory diseases – mostly autoimmune disorders like rheumatoid arthritis, psoriadic arthritis, Morbus Chron Bexxar®Corixa Corp. and GlaxoSmithKlineCD20murine — covalentely bound to Iodine 131 2003Non-Hodgkin’s lymphoma Xolair®Genentech, Tanox, Inc., Novartis PharmaceuticalsIgEhumanized2003Severe (allergic) asthma Avastin TMGenentechVEGFhumanized2004Metastatic colorectal cancer, non-small cell lung cancer, metastatic breast bancer TYSABRI®Biogen Idec and Elan Corp.α4 subunit of a4ß1 humanized2004Multiple Sclerosis, Chron´s disease ErbituxTMMerck KG aA / Bristol-Myers Squibb / ImClone Systems EGFRchimeric2004Colorectal cancer, head and neck cancer Vectibix ™AmgenEGFRhuman2006Metastatic colorectal carcinoma LUCENTIS™GenentechVEGF-Ahumanized Fab2006Wet Macular Degeneration Soliris®Alexion Pharmaceuticals, Inc.CD59humanized2007Paroxysmmal nocturnal hemoglobinuria CIMZIA®UCBTNF-aHumanized (Fab)2008Morbus Chron, rheumatoid arthritis SimponiTMCentocor (subsidiary of Johnson & Johnson)TNF-ahuman2009Rheumatoid & psoriatic arthritis, active ankylosing spondylitis 6

7. Marketed Antibody Products Company NameName of Product (1) Indications Date of FDA Approval Antibody Type (2) Ortho Biotech Orthoclone-OKT ® Organ Transplant Rejection1986M J&J/Eli Lilly ReoPro ® Acute Cardiac Conditions1994C BiogenIdec/Genentech/Roche Rituxan ® Non-Hodgkin ’ s Lymphoma 1997C BiogenIdecZevalin ™ Non-Hodgkin ’ s Lymphoma 2002M PDLI Zenapax ® Acute Transplant Rejection1997H MedImmune/Abbott Synagis ® Viral Respiratory Disease1998H Genentech/Roche Herceptin ® Avastin ® Breast Cancer Colorectal Cancer 1998 2004 HHHH J & J Remicade ® Crohn ’ s, Rheumatoid Arthritis 1998C Novartis Simulect ® Acute Transplant Rejection1998C WyethMylotarg ™ Acute Myleoid Leukemia2000H Schering /ILEX Oncology Campath ® Chronic Lymphocytic Leukemia 2001H Abbott/CATHumira ™ Rheumatoid Arthritis2002PD Novartis/Genentech/Tanox Xolair ® Asthma2003H Genentech/XomaRaptiva ™ Psoriasis2003H Corixa/GlaxoSmithKline Bexxar ® Non-Hodgkin ’ s Lymphoma 2003M BMS/ImClone SystemsErbitux ™ Colorectal Cancer2004C 7

8. 8

9. Why only 23 approved monoclonals after 35 years? How do you make human antibodies –Transgenic animals –Phage display –Ribosome RNA display –Microbial cell / yeast display Development takes time Approval takes time –We want safe drugs 9

10. Sponsor company Generic nameUS trade name mAb typeTherapeutic category US approval Johnson & Johnson Muromonab-CD3 Orthoclone OKT3 MurineImmunological19.06.1986 CentocorAbciximabReoProChimericHemostasis22.12.1994 Biogen IDECRituximabRituxanChimericAntineoplastic26.11.1997 Protein Design Daclizumab Labs | ZenapaxHumanizedImmunological10.12.1997 NovartisBasiliximabSimulectChimericImmunological12.05.1998 MedImmunePalivizumabSynagisHumanizedAnti-infective19.06.1998 CentocorInfliximabRemicadeChimericImmunological24.08.1998 GenentechTrastuzumabHerceptinHumanizedAntineoplastic25.09.1998 Wyeth Gemtuzumab ozogamicin MylotargHumanizedAntineoplastic17.05.2000 Millennium/IL EX AlemtuzumabCampathHumanizedAntineoplastic07.05.2001 Biogen IDEC Ibritumomab tiuxetan ZevalinMurineAntineoplastic19.02.2002 AbbottAdalimumabHumiraHumanImmunological31.12.2002 GenentechOmalizumabXolairHumanizedImmunological20.06.2003 CorixaTositumomab-I131BEXXARMurineAntineoplastic27.06.2003 GenentechEfalizumabRaptivaHumanizedImmunological27.10.2003 Imclone Systems CetuximabErbituxChimericAntineoplastic12.02.2004 GenentechBevacizumabAvastinHumanizedAntineoplastic26.02.2004 MAbs approved for human therapy EGF-R colon cancer HER-2/neu (EGF2) breast cancer CD33 leukemia (AML) VEGF colon cancer IgE asthma Respiratory infection Synciitial Virus IL-2, immunosupressant Stroke Transplantation Lymphoma Transplantation Arthritis Lymphoma Arthritis Lymphoma Psoriasis Leukemia 10

11. Why not mouse antibodies? Made relatively easily by hybridoma technology Main problem is “Human Anti-Mouse Antibodies” HAMA -The mouse antibodies are seen as foreign and induce an immune response -Anti-mouse antibodies are made -Endanger the patient -Enhance the clearance of the Ab -Reduce the therapeutic effect -Create immune complexes -Mouse antibodies do not activate human effector functions -Complement -Antibody mediated cytotoxicity 11

12. Anticorpi monoclonali murini mediante la tecnologia dell’ibridoma Anticorpi monoclonali CDR murini chimericiumanizzati umani Regione V murina murini - Endanger the patient - enhance the clearance of the Ab - reduce its therapeutic effect. Human immune response to mouse antibodies HAMA = H uman A nti- M ouse A ntibodies 12

13. The general trend for antibody approvals Murine monoclonals Chimeric monoclonals Humanized monoclonals Human monoclonals Mouse CDRs chimericHumanized Human Mouse V regions mouse

14. Sponsor company Generic nameUS trade name mAb typeTherapeutic category US approval Johnson & Johnson Muromonab-CD3 Orthoclone OKT3 MurineImmunological19.06.1986 CentocorAbciximabReoProChimericHemostasis22.12.1994 Biogen IDECRituximabRituxanChimericAntineoplastic26.11.1997 Protein Design Daclizumab Labs | ZenapaxHumanizedImmunological10.12.1997 NovartisBasiliximabSimulectChimericImmunological12.05.1998 MedImmunePalivizumabSynagisHumanizedAnti-infective19.06.1998 CentocorInfliximabRemicadeChimericImmunological24.08.1998 GenentechTrastuzumabHerceptinHumanizedAntineoplastic25.09.1998 Wyeth Gemtuzumab ozogamicin MylotargHumanizedAntineoplastic17.05.2000 Millennium/IL EX AlemtuzumabCampathHumanizedAntineoplastic07.05.2001 Biogen IDEC Ibritumomab tiuxetan ZevalinMurineAntineoplastic19.02.2002 AbbottAdalimumabHumiraHumanImmunological31.12.2002 GenentechOmalizumabXolairHumanizedImmunological20.06.2003 CorixaTositumomab-I131BEXXARMurineAntineoplastic27.06.2003 GenentechEfalizumabRaptivaHumanizedImmunological27.10.2003 Imclone Systems CetuximabErbituxChimericAntineoplastic12.02.2004 GenentechBevacizumabAvastinHumanizedAntineoplastic26.02.2004 MAbs approved for human therapy EGF-R colon cancer HER-2/neu (EGF2) breast cancer CD33 leukemia (AML) VEGF colon cancer IgE asthma Respiratory infection Synciitial Virus IL-2, immunosupressant Stroke Transplanation Lymphoma Transplanation Arthritis Lymphoma Arthritis Lymphoma Psoriasis Leukemia 14

15. Chimera 15

16. 16

17. How to make a chimeric antibody Murine Chimeric Human

18. Anticorpi monoclonali murini mediante la tecnologia dell’ibridoma Anticorpi chimerici (70% DNA umano solo la regione V è murina) Anticorpi monoclonali CDR murini chimericiumanizzati umani Regione V murina murini 18 #3 rituximab (Rituxan®), with sales of USD 7.285 b; #7 infliximab (Remicade®), with sales of USD 6.139 b;

19. Humanized

20. What about a humanized antibody? Murine Pick human gene that most resembles the murine gene Informatically insert the murine CDRs into the human V gene Synthesize the humanized gene Chimeric gene

21. 21 Anticorpi umanizzati (95% DNA umano solo i CDR3 sono murini)

22. Nature Reviews Cancer 1; 118-129 (2001) IMPROVING THE EFFICACY OF ANTIBODY-BASED CANCER THERAPIES 22

23. Sponsor company Generic nameUS trade name mAb typeTherapeutic category US approval Johnson & Johnson Muromonab-CD3 Orthoclone OKT3 MurineImmunological19.06.1986 CentocorAbciximabReoProChimericHemostasis22.12.1994 Biogen IDECRituximabRituxanChimericAntineoplastic26.11.1997 Protein Design Daclizumab Labs | ZenapaxHumanizedImmunological10.12.1997 NovartisBasiliximabSimulectChimericImmunological12.05.1998 MedImmunePalivizumabSynagisHumanizedAnti-infective19.06.1998 CentocorInfliximabRemicadeChimericImmunological24.08.1998 GenentechTrastuzumabHerceptinHumanizedAntineoplastic25.09.1998 Wyeth Gemtuzumab ozogamicin MylotargHumanizedAntineoplastic17.05.2000 Millennium/IL EX AlemtuzumabCampathHumanizedAntineoplastic07.05.2001 Biogen IDEC Ibritumomab tiuxetan ZevalinMurineAntineoplastic19.02.2002 AbbottAdalimumabHumiraHumanImmunological31.12.2002 GenentechOmalizumabXolairHumanizedImmunological20.06.2003 CorixaTositumomab-I131BEXXARMurineAntineoplastic27.06.2003 GenentechEfalizumabRaptivaHumanizedImmunological27.10.2003 Imclone Systems CetuximabErbituxChimericAntineoplastic12.02.2004 GenentechBevacizumabAvastinHumanizedAntineoplastic26.02.2004 MAbs approved for human therapy EGF-R colon cancer HER-2/neu (EGF2) breast cancer CD33 leukemia (AML) VEGF colon cancer IgE asthma Respiratory infection Synciitial Virus IL-2, immunosupressant Stroke Transplanation Lymphoma Transplanation Arthritis Lymphoma Arthritis Lymphoma Psoriasis Leukemia 23

24. 24

25. 25

26. 26 2012 global sales of antibody-based therapeutics exceeded USD50 billion 6 of the top 12 selling drugs in 2012 were antibody-based. The 6 products are: #1 adalimumab ( Humira®), with sales of USD9.265 b #3 rituximab (Rituxan®), with sales of USD 7.285 b; #5 trastuzumab ( Herceptin® ), with sales of USD 6.397 b; #7 infliximab (Remicade®), with sales of USD 6.139 b; #8 bevacizumab (Avastin®), with sales of USD 6.260 b; #11 etanercept (Enbrel®) with sales of USD 4.236 b.

27. Although the MRC is not primarily concerned with making money from inventions, the scale of LMB’s contribution to the field of therapeutic antibodies has led to significant revenues back to the MRC. For example, from 2000 to 2006 the MRC received approximately £84M from humanized antibodies £127M from human antibodies (share sales and royalties), of which the LMB and the inventors and associated co-workers have received a share through the MRC’s Awards to Inventors Scheme.

28. Sponsor company Generic nameUS trade name mAb typeTherapeutic category US approval Johnson & Johnson Muromonab-CD3 Orthoclone OKT3 MurineImmunological19.06.1986 CentocorAbciximabReoProChimericHemostasis22.12.1994 Biogen IDECRituximabRituxanChimericAntineoplastic26.11.1997 Protein Design Daclizumab Labs | ZenapaxHumanizedImmunological10.12.1997 NovartisBasiliximabSimulectChimericImmunological12.05.1998 MedImmunePalivizumabSynagisHumanizedAnti-infective19.06.1998 CentocorInfliximabRemicadeChimericImmunological24.08.1998 GenentechTrastuzumabHerceptinHumanizedAntineoplastic25.09.1998 Wyeth Gemtuzumab ozogamicin MylotargHumanizedAntineoplastic17.05.2000 Millennium/IL EX AlemtuzumabCampathHumanizedAntineoplastic07.05.2001 Biogen IDEC Ibritumomab tiuxetan ZevalinMurineAntineoplastic19.02.2002 AbbottAdalimumabHumiraHumanImmunological31.12.2002 GenentechOmalizumabXolairHumanizedImmunological20.06.2003 CorixaTositumomab-I131BEXXARMurineAntineoplastic27.06.2003 GenentechEfalizumabRaptivaHumanizedImmunological27.10.2003 Imclone Systems CetuximabErbituxChimericAntineoplastic12.02.2004 GenentechBevacizumabAvastinHumanizedAntineoplastic26.02.2004 MAbs approved for human therapy EGF-R colon cancer HER-2/neu (EGF2) breast cancer CD33 leukemia (AML) VEGF colon cancer IgE asthma Respiratory infection Synciitial Virus IL-2, immunosupressant Stroke Transplanation Lymphoma Transplanation Arthritis Lymphoma Arthritis Lymphoma Psoriasis Leukemia 28

29. Anticorpi monoclonali murini mediante la tecnologia dell’ibridoma Anticorpi chimerici (70% DNA umano) Anticorpi umanizzati (95% DNA umano solo i CDR3 sono murini) ANTICORPI COMPLETAMENTE UMANI Anticorpi monoclonali CDR murini chimericiumanizzati umani Regione V murina murini 29

30. 30

31. Murine monoclonals Made using hybridoma technology - everything is mouse Chimeric antibodies All constant regions are human, variable are mouse: 70% human Humanized antibodies Only the antibody binding loops (CDRs - complementarity determining regions) are mouse, the rest is human: 95% human Human antibodies Everything is human Monoclonal antibodies 31

32. What they really look like Mouse Chimeric Human Humanized 32

33. 33

34. MAbs in Action Hybridoma ZenapaxRituxan HerceptinRemicadeSynagisSimulect Mylotarg Campath Chimerization Humanization Phage Display Transgenic Mice ReoPro 2001 2000 1998 1997 1994 1986 1985 1975 OKT3 2002 Zevalin Yeast Display 2003 Humira XolairBexxar Ribosome Display TechnologyMAb Approved       2004 AvastinErbitux  34

35. Making human antibodies Transgenic animals Phage display Ribosome RNA display Microbial cell / yeast display 35

36. Transgenic Mouse H  VH1VH1V H 65VH2VH2D H 1-------27J H 1-----6 CC 36

37. 37

38. 38

39. 39

40. ( 80 kb ) Grafting human heavy- and light-chain miniloci into mice & producing human mAb 40

41. What they really look like Mouse Chimeric Human Humanized 41

42. XenoMouse ® -  Strains IgG 1  IgG 2  IgG 4  IgG 1 IgG 1 IgG 2 IgG 2 IgG 4 IgG 4 IgG 1 IgG 1 IgG 2 IgG 2 IgG 4 IgG 4 42

43. The TC Mouse™ was developed through the use of Kirin's proprietary microcell mediated chromosome transfer (MMCT) technology which has the capability to introduce large portions of the human chromosome into mice. The TC Mouse™ carries human chromosomal fragments which contain the complete human immunoglobulin loci. Therefore, the TC Mouse™ Technology is distinguished from other antibody producing transgenic mice by being a Transchromosomic mouse, carrying the full human immunoglobulin repertoire. 43

44. Ci sono 3 ditte che dispongono di topi –mAb-human Ci sono più di 35 anticorpi umani in trials clinici avanzati 44

45. Transgenic mice These produce human antibodies following immunization The creation of monoclonal antibodies is the same as normal mice using hybridoma technology This technology is not used to produce large amounts of antibody Antibody production is still required after selection 45

46. 46

47. 47

48. Making human antibodies Transgenic animals Phage display Ribosome RNA display Microbial cell / yeast display 48

49. 49