Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

  • Moon, Seung Kee (Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm.) ;
  • Park, So Ra (Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm.) ;
  • Park, Ami (Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm.) ;
  • Oh, Hyun Mi (Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm.) ;
  • Shin, Hyun Jung (Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm.) ;
  • Jeon, Eun Ju (Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm.) ;
  • Kim, Seiwhan (Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm.) ;
  • Park, Hyun June (Graduate Program of Bioengineering, Seoul National University) ;
  • Yeon, Young Joo (The Institute of Molecular Biology and Genetics, Seoul National University) ;
  • Yoo, Young Je (Graduate Program of Bioengineering, Seoul National University)
  • Received : 2015.09.03
  • Accepted : 2015.10.26
  • Published : 2016.03.31
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Abstract

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their antiproliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity ($IC_{50}$: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity ($K_D$: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCIN82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.

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References

  1. Back, H., Suk, K.H., Kim, Y.H., and Cha, S.H. (2002). An improved helper phage system for efficient isolation of specific antibody molecules in phage display. Nucleic Acids Res. 30, e18-e26. https://doi.org/10.1093/nar/30.5.e18
  2. Carter, P., Presta, L., Gorman, C.M., Ridgway, J.B., Henner, D., Wong, W.L., Rowland, A.M., Kotts, C., Carver, M.E., and Shepard, H.M. (1992). Humanization of an anti-p185 HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. USA 89, 4285-4289. https://doi.org/10.1073/pnas.89.10.4285
  3. Chatellier, J., Van Regenmortel, M.H., Vernet, T., and Altschuh, D. (1996). Functional mapping of conserved residues located at the VL and VH domain interface of a Fab. J. Mol. Biol. 264, 1-6. https://doi.org/10.1006/jmbi.1996.0618
  4. Chen, Y., Wiesmann, C., Fuh, G., Li, B., Christinger, H.W., McKay, P., de Vos, A.M., and Lowman, H.B. (1999). Selection and analysis of an optimized anti-VEGF antibody crystal structure of an affinity-matured Fab in complex with antigen. J. Mol. Biol. 293, 865-881. https://doi.org/10.1006/jmbi.1999.3192
  5. Genentech, Inc. (2005). Humanized anti-ErbB2 antibodies and treatment with anti-ErbB2 antibodies. US6949245 (US Patent).
  6. Gerstner, R.B., Carter, P., and Lowman, H.B. (2002). Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody. J. Mol. Biol. 321, 851-862. https://doi.org/10.1016/S0022-2836(02)00677-0
  7. Gong, S.J., Jin, C.J., Rha, S.Y., and Chung, H.C. (2004). Growth inhibitory effects of trastuzumab and chemotherapeutic drugs in gastric cancer cell lines. Cancer Lett. 214, 215-224. https://doi.org/10.1016/j.canlet.2004.04.029
  8. Hawkins, R.E., Russell, S.J., Baier, M., and Winter, G. (1993). The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme. J. Mol. Biol. 234, 958-964. https://doi.org/10.1006/jmbi.1993.1650
  9. Hudziak, R.M., Lewis, G.D., Winget, M., Fendly, B.M., Shepard, H.M., and Ullrich, A. (1989). p185 HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol. Cell. Biol. 9, 1165-1172. https://doi.org/10.1128/MCB.9.3.1165
  10. Johnsson, B., Löfås, S., and Lindquist, G.. (1991). Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Analyt. Biochem. 198, 268-277. https://doi.org/10.1016/0003-2697(91)90424-R
  11. Kabat, E.A., and Wu, T.T. (1991). Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL gene, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J. Immunol. 147,1709-1719.
  12. Kelley, R.F., and O'Connell, M.P. (1993). Thermodynamic analysis of an antibody functional epitope. Biochemistry 32, 6828-6835. https://doi.org/10.1021/bi00078a005
  13. Kim, J.W., Kim, H.P., Im, S.A., Kang, S., Hur, H.S., Yoon, Y.K., Oh, D.Y., Kim, J.H., Lee, D.S., Kim, T.Y., and Bang, Y.J. (2008). The growth inhibitory effect of lapatinib, a dual inhibitor of EGFR and HER2 tyrosine kinase, in gastric cancer cell lines. Cancer Lett. 272, 296-306. https://doi.org/10.1016/j.canlet.2008.07.018
  14. Lesley, J., Hascall V.C., Tammi, M., and Hyman, R. (2000). Hyaluronan binding by cell surface CD44. J. Biol. Chem. 275, 26967-26975.
  15. Lowe, D., Wilkinson T., and Vaughan, T.J., (2012). Affinity maturation approaches for antibody lead optimization. in Antibody Drug Discovery, Wood C.R., (London, UK : Imperial College Press). pp. 85-120.
  16. Macdonald, R.A., Hosking, C.S., and Jones, C.L. (1988). The measurement of relative antibody affinity by ELISA using thiocyanate elution. J. Immunol. Methods. 106,191-194. https://doi.org/10.1016/0022-1759(88)90196-2
  17. Mayumi, O., and Michihiko, K. (2006). Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to Gefitinib and other EGFR-targeting drugs. Clin. Cancer Res. 12, 7242-7251. https://doi.org/10.1158/1078-0432.CCR-06-0646
  18. Nicolas, W., Véronique, D.H., and Martine, J.P. (2008). HER2- positive breast cancer: from trastuzumab to innovatory anti-HER2 strategies. Clin. Breast Cancer 8, 38-49. https://doi.org/10.3816/CBC.2008.n.002
  19. O'Brien, N.A., Browne, B.C., Chow, L., Wang, Y., Ginther, C., Arboledal, J., Duffy, M.J., Crown, J., O'Donovan, N., and Slamon, J.D. (2010). Activated phosphoinositide 3-kinase/AKT signaling confers resistance to Trastuzumab but not Lapatinib. Mol. Cancer Ther. 9, 1489-2211. https://doi.org/10.1158/1535-7163.MCT-09-1171
  20. Oh, M.Y., oung, Joo, H.Y., Hur, B.U., Jeong, Y.H., and Cha, S.H. (2007). Enhancing phage display of antibody fragments using gIII-amber suppression. Gene 386, 81-89. https://doi.org/10.1016/j.gene.2006.08.009
  21. Phumyen, A., Jumnainsong, A., and Leelayuwat, C. (2012). Improved binding activity of antibodies against major histocompatibility complex Class I chain-related gene A by phage display technology for cancer-targeted therapy. J. Biomed. Biotechnol. 2012,1-8.
  22. Rajpal, A., Beyaz, N., Haber, L., Cappuccilli, G., Yee, H., Bhatt, R.R., Takeuchi, T., Lerner, R.A., and Crea, R. (2005). A general method for greatly improving the affinity of antibodies by using combinatorial libraries. Proc. Natl. Acad. Sci. USA 102, 8466-8471. https://doi.org/10.1073/pnas.0503543102
  23. Romond, E.H., Perez, E.A., Bryant, J., Suman, V.J., Geyer, C.E. Jr., Davidson, N.E., Tan-Chiu, E., Martino, S., Paik, S., Kaufman, P.A., et al. (2005). Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N. Engl. J. Med. 353, 1673-1684. https://doi.org/10.1056/NEJMoa052122
  24. Schier, R., McCall, A., Adams, G.P., Marshall, K.W., Merritt, H., Yim, M., Crawford, R.S., Weiner, L.M., Marks, C., and Marks, J.D. (1996). Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J. Mol. Biol. 263, 551-567. https://doi.org/10.1006/jmbi.1996.0598
  25. Shepard, H.M., Lewis, G.D., Sarup, J.C., Fendly, B.M., Maneval, D., Mordenti, J., Figari, I., Kotts, C.E., Palladino, M.A. Jr, Ullrich, A., et al. (1991). Monoclonal antibody therapy of human cancer: taking the HER2 protooncogene to the clinic. J. Clin. Immunol. 11, 117-127. https://doi.org/10.1007/BF00918679
  26. Sidhu, S.S, Lowman, H.B., Cunningham, B.C., and Wells, J.A. (2000). Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333-363. https://doi.org/10.1016/S0076-6879(00)28406-1
  27. Sliwkowski, M.X., Lofgren, J.A., Lewis, G.D., Hotaling, T.E., Fendly, B.M., and Fox, J.A. (1999). Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin. Oncol. 26, 60-70.
  28. Song, S.Y., Hur, B.U., Lee, K.W., Choi, H.J., Kim, S.S., Kang, K., and Cha, S.H. (2009). Successful application of the dual-vector system II in creating a reliable phage-displayed combinatorial Fab library. Mol. Cells 27, 313-319. https://doi.org/10.1007/s10059-009-0040-0
  29. Wang, Z., Wang, Y., Li, Z., Li, J., and Dong, Z. (2000). Humanization of a mouse monoclonal antibody neutralizing TNF-alpha by guided selection. J. Immunol. Methods 241, 171-184. https://doi.org/10.1016/S0022-1759(00)00203-9
  30. Yakes, F.M., Chinratanalab, W., Ritter, C.A., King, W., Seelig, S., and Arteaga, C.L. (2002). Herceptin-induced inhibition of phosphatidylinositol- 3 kinase and Akt is required for antibodymediated effects on p27, cyclin D1, and antitumor action. Cancer Res. 62, 4132-4141.
  31. Yoon, S.O., Lee, T.S., Kim, S.J., Jang, M.H., Kang. Y.J., Park, J.H., Kim, K.S., Lee, H.S., Ryu, C.J., Gonzales, N.R., et al. (2006). Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody. J. Biol. Chem. 281, 6985-6992. https://doi.org/10.1074/jbc.M511165200

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