1. 항체의약품 개발의 최근 동향 Hyo Jeong Hong Laboratory of Immunology KRIBB

2. Humanization of Monoclonal Antibody

3. 인간화항체의 제조기술

4. Antibody Phage Display Technology – Human Monoclonal Antibodies

6. Antibody Fragments

7. ANTIBODY TYPE SPECIFICITYAPPLICATIONCOMPANYTRADE NAME FDA APPROVA L Chimeric FabGPIIb/IIIaPreventing blood clotCentocorReoPro1994 Humanized antibody IL-2 receptorPrevention of acute kidney transplant rejection Hoffmann- La Roche Zenapax1997 Chimeric antibody CD20Non-Hodgkin's B-cell lymphoma GenentechRituxan1997 Humanized antibody RSVProphylaxis of RSV infectionMedImmuneSynagis1998 Humanized antibody TNF-αCrohn Disease, Rheumatoid artritis CentocorRimicade1998 Humanized antibody Her2/neuBreast cancerGenentechHerceptin1998 Chimeric antibody CD25ImmunosupressionNovartisSimulect1998 Humanized antibody CD3ImmunosupressionWyeth-AyerstMylotarg2000 Humanized antibody CD52Chronic Lymphocytic Leukemia MillenniumCampath2001 Humanized antibody CD20Non-Hodgkin ’ s lymphomaIDECZevalin2002 Table 1. Humanized antibodies in clinical use

8. YearNameTargetApplicationCompanyAb Type 2002 HumiraTNF-a 관절염 CAT/BASFHuman 2003XolairIgE 천식 Tanox/ Genentech/ Novartis Humanized 2003RaptivaCD11a 건선 Xoma/ Genentech Humanied 2004ErbituxEGFR 전이성 대장암 ImcloneHumanized 2004AvastinVEGF 대장암유방 암 신장암 NSCL GenentechHumanized Table 1. Humanized/Human antibodies in clinical use

10. Why mAbs? Proven effective for a broad range of indications –Immunosuppression, inflammatory disease, infections and cancer Have excellent safety profiles: highly specific –Early decision by tissue cross-reactivity test (fast & clear development path)  Relatively short development cycle: 7 years vs 10-12 years –Remarkably free of adverse effects and synergistic therapeutic responses Commercially successful –High success rates and phase transition probabilities –~ 370 biotech-based drugs in development in 2000, 73 were mAbs –Worldwide Ab sales: $21b in 2010, with >90 Abs on the market

11. Forecast sales of key cancer monoclonal antibodies to 2010

12. Forecast sales of key monoclonal antibodies (other) to 2010

13. - Cloning of antibody genes from murine hybridoma - Grafting of mouse CDRs onto human H & L chains (CDR-grafting) - (Affinity maturation) - Construction of CHO cell line - Production of engineered antibody Construction of Humanized Antibody

14. Humanization Protocol of KR127 Kappa Light Chain FR1 CDR1 FR2 CDR2 KR127 DILMTQTPLILSVTIGQPASISC KSSQSLLYSNGKTYLN WLLQRPGQSPKRLIY LVSKLDS DPK12 DIVMTQTPLSLSVTPGQPASISC KSSQSLLHSDGKTYLY WYLQKPGQPPQLLIY EVSNRFS HZI DILMTQTPLSLSVTPGQPASISC KSSQSLLYSNGKTYLN WLLQKPGQSPKRLIY LVSKLDS FR3 CDR3 FR4 KR127 GVPDRFTGSGSGTDFTLKIIRVEAEDLGVYYC VQGTHFPQT FGGGTKLEIKR DPK12 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQSIQLPLT FGGGTKVEIKR HZI GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC VQGTHFPQT FGGGTKVEIKR

15. Construction of Recombinant CHO cells Vector construction & transfection G418 selection Gene amplification by MTX selection Cell subcloning Characterization of cell line productivity and stability

17. Isolation of Human Mab Construction of human Fab library (3.9 x10 7 ) - from 7 HAV-immune donors - Fd (VH1+CH1), kappa L chain (VL+CL) Biopanning against formalin-inactivated HAV (HM175) Production of whole Ig from HAV-specific Fab clones Characterization of anti-HAV MAbs - Epitope specificity - HAV-neutralizing activity (Radioimmunofocus Assay, RIFA)

18. Results Biopanning of Phage Fab Library

19. Anti-HAV Fab clones selected after the fourth round of panning ELISA (Positive/Total ) BstNI fingerprinting Selected Fab VH family VH segment VL family VL segment 22/2413/22HAV6VH4DP79VK1DPK9 22/247/22HAV9VH3DP47VK1DPK9 22/241/22HAV1VH4DP71VK1DPK9 22/241/22HAV12VH4DP63VK1DPK9

20. HAV binding Activity of Whole IgG

21. Cross-Competition Binding Assay Binding of biotin-labeled antibodies to HAV-coated microtiter wells was detected by ELISA in the presence of dilutions of competing antibodies, HAV1 ( ○ ), HAV6 ( ▽ ), HAV9 ( □ ), and HAV12 ( ◇ ). RESULT: HAV1 / HAV6, 9 / HAV12 3 epitopes are closely spaced

22. Competition Binding Assay with HAV-Neutralizing Murine MAbs

23. HAV-Neutralization Assay Virus Control CS13 50  g/ml HAV6 50  g/ml HAV9 50  g/ml HAV12 50  g/ml HAV1 50  g/ml

24. Conclusions 1.Four human MAbs were isolated from a phage- displayed antibody library from HAV-immune donors 2.They recognize closely spaced immunodominant epitopes. 3.All four MAbs showed HAV-neutralizing activities. 4.HAV6 will be useful in the prophylaxis of HAV infection.