CDR Repair: A Novel Approach to Antibody Humanization

  • Mark S. Dennis
Part of the Biotechnology: Pharmaceutical Aspects book series (PHARMASP, volume XI)


Hybridoma technology has enabled the rapid production of a large number of monoclonal antibodies with interesting biological properties. Their use in a therapeutic setting, however, can lead to the generation of a human anti-mouse antibody (HAMA) response in patients despite the high degree of sequence similarity shared between human and mouse antibodies. This has prompted efforts to make hybridoma antibodies appear more human through the construction of chimeras, (Morrison et al. 1984) and through a process known as antibody humanization (Riechmann et al. 1988; Verhoeyen et al. 1988).

The modular nature of antibodies makes the swapping of domains a relatively simple process. A chimera consisting of the mouse variable heavy (VH) and variable light (VL) domains recombinantly fused to human heavy and light constant domains is a simple way to reduce HAMA response. Yet, despite 60–75% homology to human, murine variable domains may still elicit a HAMA response.


Variable Domain Antigen Binding Variable Light Variable Heavy Parent Antibody 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Complementarity determining regions


Antigen binding fragment consisting of the light chain and the variable and first constant domains of the heavy chain


Human anti-mouse antibodies


Variable heavy domain


Variable light domain



I gratefully recognize the contributions made by the oligonucleotide synthesis and DNA sequencing groups at Genentech who have made this work possible. I also thank all current and former members of the Protein Engineering and Antibody Engineering Departments at Genentech for discussions and their contributions to the development of phage display methods, and antibody humanization technologies and especially, Henry Lowman for his support.


  1. Adams CW, Allison DE, Flagella K, Presta L, Clarke J, Dybdal N, McKeever K, Sliwkowski MX (2006) Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol Immunother 55:717–727PubMedCrossRefGoogle Scholar
  2. Andrew DP, Berlin C, Honda S, Yoshino T, Harnann A, HoIzmann B, Kilshaw PJ, Butcher EC (1994) Distinct but overlapping epitopes are involved in α4b7-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation. J Immunol 153:3847–3861PubMedGoogle Scholar
  3. Baca M, Presta LG, O’Connor SJ, Wells JA (1997) Antibody humanization using monovalent phage display. J Biol Chem 272:10678–10684PubMedCrossRefGoogle Scholar
  4. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242PubMedCrossRefGoogle Scholar
  5. Carter P, Presta LG, Gorman CM, Ridgway JBB, Henner D, Wong WLT, Rowland AM, Kotts C, Carver ME, Shepard MH (1992) Humanization of an anti-P185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 89:4285PubMedCrossRefGoogle Scholar
  6. Chothia C, Lesk AM (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol 196:901–917PubMedCrossRefGoogle Scholar
  7. Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill SJ, Air G, Sheriff S, Padlan EA, Davies D, Tulip WR, Colman PM, Spinelli S, Alzari PM, Poljak RJ (1989) Conformations of immunoglobulin hypervariable regions. Nature 342:877PubMedCrossRefGoogle Scholar
  8. Clark LA, Ganesan S, Papp S, van Vlijmen HWT (2006) Trends in antibody sequence changes during the somatic hypermutation process. J Immunol 177:333–340PubMedGoogle Scholar
  9. Eigenbrot C, Randal M, Presta LG, Carter P, Kossiakoff AA (1993) X-ray structures of the antigen-binding domains from three variants of humanized anti-P185HER2 antibody 4d5 and comparison with molecular modeling. J Mol Biol 229:969–995PubMedCrossRefGoogle Scholar
  10. Foote J, Winter G (1992) Antibody framework residues affecting the conformation of hypervariable loops. J Mol Biol 224:487–499PubMedCrossRefGoogle Scholar
  11. Gallop MA, Barrett RW, Dower WJ, Fodor SPA, Gordon EM (1994) Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries. J Med Chem 37:1233–1251PubMedCrossRefGoogle Scholar
  12. Honegger A (2007) AHo’s amazing atlas of antibody anatomy. Assessed June 2007
  13. Hwang WYK, Almagro JC, Buss TN, Tan P, Foote J (2005) Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods 36:35–42PubMedCrossRefGoogle Scholar
  14. Johnson G, Wu TT (2001) Kabat database and its applications: future directions. Nucleic Acids Res 29:205–206PubMedCrossRefGoogle Scholar
  15. Jones PT, Dear PH, Foote J, Neuberger MS, Winter G (1986) Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321:522–525PubMedCrossRefGoogle Scholar
  16. Kabat EA, Wu TT (1971) Attempts to locate complementarity determining residues in the variable positions of light and heavy chains. Ann N Y Acad Sci 190:382–393PubMedCrossRefGoogle Scholar
  17. Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C (1991) Sequences of proteins of immunological interest. Public Health Service, National Institutes of Health, BethesdaGoogle Scholar
  18. Kashmiri SVS, Pascalis RD, Gonzales NR, Schlom J (2005) SDR grafting – a new approach to antibody humanization. Methods 36:25–34PubMedCrossRefGoogle Scholar
  19. Kelsen J, Agnholt J, Falborg L, Nieslen JT, Romer JL, Hoffmann HJ, Dahlerup JF (2004) 111Indium-labelled human gut-derived T cells from healthy subjects with strong in vitro adhesion to MAdCAM-1 show no detectable homing to the gut in vivo. Clin Exp Immunol 138:66–74PubMedCrossRefGoogle Scholar
  20. Kettleborough CA, Saldanha J, Heath VJ, Morrison CJ, Bendig MM (1991) Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation. Protein Eng 4:773PubMedCrossRefGoogle Scholar
  21. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wolle J, Pluckthun A, Virnekas B (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57–86PubMedCrossRefGoogle Scholar
  22. Lee UH, Son JH, Lee JJ, Kwon B, Park JW, Kwon BS (2004) Humanization of antagonistic anti-human 4-1bb monoclonal antibody using a phage-displayed combinatorial library. J Immunother 27:201–210PubMedCrossRefGoogle Scholar
  23. Li B, Fuh G, Meng G, Xin X, Gerritsen ME, Cunningham B, de Vos AM (2000) Receptor-selective variants of human vascular endothelial growth factor. J Biol Chem 275:29823–29828PubMedCrossRefGoogle Scholar
  24. MacCallum RM, Martin ACR, Thornton JT (1996) Antibody–antigen interactions: contact analysis and binding site topography. J Mol Biol 262:732–745PubMedCrossRefGoogle Scholar
  25. Maynard JA, Maassen CBM, Leppla SH, Brasky K, Patterson JL, Iversone BL, Georgiou G (2002) Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat Biotechnol 20:597–601PubMedCrossRefGoogle Scholar
  26. Morrison SL, Johnson MJ, Herzenberg LA, Oi VT (1984) Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci U S A 81:6851–6855PubMedCrossRefGoogle Scholar
  27. Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang LH, Gorlatov S, Mehlhop E, Marri A, Chung KM, Ebel GD, Kramer LD, Fremont DHF, Diamond MS (2005) Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 11:522–530PubMedCrossRefGoogle Scholar
  28. Padlan EA (1994) Anatomy of the antibody molecule. Mol Immunol 31:169–217PubMedCrossRefGoogle Scholar
  29. Presta LG, Lahr SJ, Shields RL, Porter JP, Gorman CM, Fendly BM, Jardieu PM (1993) Humanization of an antibody directed against IgE. J Immunol 151:2623–2632PubMedGoogle Scholar
  30. Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, Krummen L, Winkler M, Ferrara N (1997) Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57:4593–4599PubMedGoogle Scholar
  31. Presta LG, Sims P, Meng YG, Moran P, Bullens S, Bunting S, Schoenfeld J, Lowe D, Lai J, Rancatore P, Iverson M, Lim M, Chisholm V, Kelley RF, Riederer M, Kirchhofer D (2001) Generation of a humanized, high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic. Thromb Haemost 85:379–389PubMedGoogle Scholar
  32. Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF, Duncan JF, Avdalovic NM, Levitt M, Junghans RP, Waldmann TA (1989) A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci U S A 86:10029–10033PubMedCrossRefGoogle Scholar
  33. Rader C, Ritter G, Nathan S, Elia M, Gout I, Jungbluth AA, Cohen LS, Welt S, Old LJ, Barbas CF III (2000) The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies. J Biol Chem 275:13668–13676PubMedCrossRefGoogle Scholar
  34. Riechmann L, Clark M, Waldmann H, Winter G (1988) Reshaping human antibodies for therapy. Nature 332:323–327PubMedCrossRefGoogle Scholar
  35. Schlapschy M, Gruber H, Gresch O, Schafer C, Renner C, Pfreundschuh M, Skerra A (2004) Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin’s lymphoma using an in vitro evolution approach. Protein Eng Des Sel 17:847–860PubMedCrossRefGoogle Scholar
  36. Sidhu SS, Li B, Chen Y, Fellouse FA, Eigenbrot C, Fuh G (2004) Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J Mol Biol 338:229–310CrossRefGoogle Scholar
  37. Tan P, Mitchell DA, Buss TN, Holmes MA, Anasetti C, Foote J (2002) “Superhumanized” antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28. J Immunol 169:1119–1125PubMedGoogle Scholar
  38. Tsurushita N, Hinton PR, Kumar S (2005) Design of humanized antibodies: from anti-Tac to Zenapax. Methods 36:69–83PubMedCrossRefGoogle Scholar
  39. Verhoeyen M, Milstein C, Winter G (1988) Reshaping human antibodies: grafting an antilysozyme activity. Science 239:1534–1536PubMedCrossRefGoogle Scholar
  40. Wang X-BW, Zhou B, Yin C-C, Lin Q, Huang H-L (2004) A new approach for rapidly reshaping single-chain antibody in vitro by combining DNA shuffling with ribosome display. J Biochem 136:19–28PubMedCrossRefGoogle Scholar
  41. Werther WA, Gonzalez TN, O’Connor SJ, McCabe S, Chan B, Hotaling T, Champe M, Fox JA, Jardieu PM, Berman PW, Presta LG (1996) Humanization of an anti-lymphocyte function-associated antigen (LFA-1) monoclonal antibody and reengineering of the humanized antibody for binding to rhesus LFA-1. J Immunol 157:4986–4995PubMedGoogle Scholar
  42. Wu TT, Kabat EA (1970) An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J Exp Med 132:211–250PubMedCrossRefGoogle Scholar
  43. Wu H, Nie Y, Huse WD, Watkins JD (1999) Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues. J Mol Biol 294:151–162PubMedCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2010

Authors and Affiliations

  1. 1.Department of Antibody EngineeringGenentech, Inc.South San FranciscoUSA

Personalised recommendations