Technologies for the Generation of Human Antibodies

  • Ramesh R. Bhatt
  • John S. Haurum
  • C. Geoffrey Davis


Over the course of the last 15 years antibodies as drugs have come into their own—there are now 26 therapeutic antibodies on the market in the United States. With the passing of time, new technological developments together with competition for finite markets have continually raised the bar for the specifications of newly introduced antibodies. Our intent in this review is to provide a historical perspective on the technologies that have generated the fully human antibody drugs currently on the market as well as to impart a sense of excitement for the technologies in development that will provide the antibody drugs of the future.


Light Chain Phage Display Somatic Hypermutation Phage Display Library Antibody Library 
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.


  1. Andersen PS, Haahr-Hansen M et al (2007) Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen. Mol Immunol 44(4):412–422PubMedCrossRefGoogle Scholar
  2. Bankovich AJ, Raunser S et al (2007) Structural insight into pre-B cell receptor function. Science 316(5822):291–294PubMedCrossRefGoogle Scholar
  3. Barbas CF 3rd, Bain JD et al (1992a) Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Proc Natl Acad Sci U S A 89(10):4457–4461PubMedCrossRefGoogle Scholar
  4. Barbas CF 3rd, Crowe JE Jr et al (1992b) Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc Natl Acad Sci U S A 89(21):10164–10168PubMedCrossRefGoogle Scholar
  5. Barbas CF 3rd, Kang AS et al (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88(18):7978–7982PubMedCrossRefGoogle Scholar
  6. Bass S, Greene R et al (1990) Hormone phage: an enrichment method for variant proteins with altered binding properties. Proteins 8(4):309–314PubMedCrossRefGoogle Scholar
  7. Beerli RR, Bauer M et al (2008) Isolation of human monoclonal antibodies by mammalian cell display. Proc Natl Acad Sci U S A 105(38):14336–14341PubMedCrossRefGoogle Scholar
  8. Beerli RR, Bauer M et al (2009) Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against influenza A M2 protein. Virol J 6:224PubMedCrossRefGoogle Scholar
  9. Boder ET, Midelfort KS et al (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci U S A 97(20):10701–10705PubMedCrossRefGoogle Scholar
  10. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15(6):553–557PubMedCrossRefGoogle Scholar
  11. Brezinschek HP, Brezinschek RI et al (1995) Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J Immunol 155(1):190–202PubMedGoogle Scholar
  12. Bruggemann M, Caskey HM et al (1989) A repertoire of monoclonal antibodies with human heavy chains from transgenic mice. Proc Natl Acad Sci U S A 86(17):6709–6713PubMedCrossRefGoogle Scholar
  13. Burton DR, Barbas CF 3rd et al (1991) A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci U S A 88(22):10134–10137PubMedCrossRefGoogle Scholar
  14. Chen W, Zhu Z et al (2010) A large human domain antibody library combining heavy and light chain CDR3 diversity. Mol Immunol 47(4):912–921PubMedCrossRefGoogle Scholar
  15. Chen W, Zhu Z et al (2008) Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain. J Mol Biol 382(3):779–789PubMedCrossRefGoogle Scholar
  16. Collarini EJ, Lee FE et al (2009) Potent high-affinity antibodies for treatment and prophylaxis of respiratory syncytial virus derived from B cells of infected patients. J Immunol 183(10):6338–6345PubMedCrossRefGoogle Scholar
  17. Cwirla SE, Peters EA et al (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci U S A 87(16):6378–6382PubMedCrossRefGoogle Scholar
  18. de Haard HJ, van Neer N et al (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274(26):18218–18230PubMedCrossRefGoogle Scholar
  19. de Kruif J, Kramer A et al (2010) Generation of stable cell clones expressing mixtures of human antibodies. Biotechnol Bioeng 106(5):741–750PubMedCrossRefGoogle Scholar
  20. Els Conrath K, Lauwereys M et al (2001) Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem 276(10):7346–7350PubMedCrossRefGoogle Scholar
  21. Feldhaus MJ, Siegel RW et al (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21(2):163–170PubMedCrossRefGoogle Scholar
  22. Fellouse FA, Esaki K et al (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373(4):924–940PubMedCrossRefGoogle Scholar
  23. Fellouse FA, Wiesmann C et al (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci U S A 101(34):12467–12472PubMedCrossRefGoogle Scholar
  24. Foote J, Eisen HN (1995) Kinetic and affinity limits on antibodies produced during immune responses. Proc Natl Acad Sci U S A 92(5):1254–1256PubMedCrossRefGoogle Scholar
  25. Gallo ML, Ivanov VE et al (2000) The human immunoglobulin loci introduced into mice: V(D) and J gene segment usage similar to that of adult humans. Eur J Immunol 30(2):534–540PubMedCrossRefGoogle Scholar
  26. Gao C, Mao S et al (2002) A method for the generation of combinatorial antibody libraries using pIX phage display. Proc Natl Acad Sci U S A 99(20):12612–12616PubMedCrossRefGoogle Scholar
  27. Geurts AM, Cost GJ et al (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325(5939):433PubMedCrossRefGoogle Scholar
  28. Grandea AG 3rd, Olsen OA et al (2010) Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses. Proc Natl Acad Sci U S A 107(28):12658–12663PubMedCrossRefGoogle Scholar
  29. Green LL, Hardy MC et al (1994) Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet 7(1):13–21PubMedCrossRefGoogle Scholar
  30. Green LL, Jakobovits A (1998) Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. J Exp Med 188(3):483–495PubMedCrossRefGoogle Scholar
  31. Hamers-Casterman C, Atarhouch T et al (1993) Naturally occurring antibodies devoid of light chains. Nature 363(6428):446–448PubMedCrossRefGoogle Scholar
  32. Hanes J, Jermutus L et al (1998) Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc Natl Acad Sci U S A 95(24):14130–14135PubMedCrossRefGoogle Scholar
  33. Hanes J, Schaffitzel C et al (2000) Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol 18(12):1287–1292PubMedCrossRefGoogle Scholar
  34. Harriman WD, Collarini EJ et al (2009) Antibody discovery via multiplexed single cell characterization. J Immunol Methods 341(1–2):135–145PubMedCrossRefGoogle Scholar
  35. He M, Taussig MJ (1997) Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res 25(24):5132–5134PubMedCrossRefGoogle Scholar
  36. Hinton PR, Johlfs MG et al (2004) Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem 279(8):6213–6216PubMedCrossRefGoogle Scholar
  37. Hoet RM, Cohen EH et al (2005) Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat Biotechnol 23(3):344–348PubMedCrossRefGoogle Scholar
  38. Hoogenboom HR, Griffiths AD et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19(15):4133–4137PubMedCrossRefGoogle Scholar
  39. Huse WD, Sastry L et al (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246(4935):1275–1281PubMedCrossRefGoogle Scholar
  40. Ishida I, Tomizuka K et al (2002) TransChromo mouse. Biotechnol Genet Eng Rev 19:73–82PubMedGoogle Scholar
  41. Jakobovits A, Amado RG et al (2007) From XenoMouse® technology to panitumumab, the first fully human antibody product from transgenic mice. Nat Biotechnol 25(10):1134–1143PubMedCrossRefGoogle Scholar
  42. Janssens R, Dekker S et al (2006) Generation of heavy-chain-only antibodies in mice. Proc Natl Acad Sci U S A 103(41):15130–15135PubMedCrossRefGoogle Scholar
  43. Jespers L, Schon O et al (2004) Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22(9):1161–1165PubMedCrossRefGoogle Scholar
  44. Jespers LS, Roberts A et al (1994) Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Biotechnology (N Y) 12(9):899–903CrossRefGoogle Scholar
  45. Kantor AB, Merrill CE et al (1995) Development of the antibody repertoire as revealed by single-cell PCR of FACS-sorted B-cell subsets. Ann N Y Acad Sci 764:224–227PubMedCrossRefGoogle Scholar
  46. Knappik A, Ge L et al (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296(1):57–86PubMedCrossRefGoogle Scholar
  47. Kopsidas G, Carman RK et al (2007) RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution. BMC Biotechnol 7:18PubMedCrossRefGoogle Scholar
  48. Kopsidas G, Roberts AS et al (2006) In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation. Immunol Lett 107(2):163–168PubMedCrossRefGoogle Scholar
  49. Kuroiwa Y, Kasinathan P et al (2002) Cloned transchromosomic calves producing human immunoglobulin. Nat Biotechnol 20(9):889–894PubMedCrossRefGoogle Scholar
  50. Lazar GA, Dang W et al (2006) Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A 103(11):4005–4010PubMedCrossRefGoogle Scholar
  51. Lee CV, Liang WC et al (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 340(5):1073–1093PubMedCrossRefGoogle Scholar
  52. Leung DW, Chen E et al (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1(1):11–15Google Scholar
  53. Ling MM (2003) Large antibody display libraries for isolation of high-affinity antibodies. Comb Chem High Throughput Screen 6(5):421–432PubMedGoogle Scholar
  54. Lloyd C, Lowe D et al (2009) Modelling the human immune response: performance of a 1011 human antibody repertoire against a broad panel of therapeutically relevant antigens. Protein Eng Des Sel 22(3):159–168PubMedCrossRefGoogle Scholar
  55. Lonberg N, Taylor LD et al (1994) Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 368(6474):856–859PubMedCrossRefGoogle Scholar
  56. Lowman HB, Bass SH et al (1991) Selecting high-affinity binding proteins by monovalent phage display. Biochemistry 30(45):10832–10838PubMedCrossRefGoogle Scholar
  57. Marks JD, Griffiths AD et al (1992) By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (N Y) 10(7):779–783CrossRefGoogle Scholar
  58. Marks JD, Hoogenboom HR et al (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222(3):581–597PubMedCrossRefGoogle Scholar
  59. Martin A, Scharff MD (2002) Somatic hypermutation of the AID transgene in B and non-B cells. Proc Natl Acad Sci U S A 99(19):12304–12308PubMedCrossRefGoogle Scholar
  60. Mattheakis LC, Bhatt RR et al (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A 91(19):9022–9026PubMedCrossRefGoogle Scholar
  61. McCafferty J, Griffiths AD et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348(6301):552–554PubMedCrossRefGoogle Scholar
  62. Melchers F (1999) Fit for life in the immune system? Surrogate L chain tests H chains that test L chains. Proc Natl Acad Sci U S A 96(6):2571–2573PubMedCrossRefGoogle Scholar
  63. Mendez MJ, Green LL et al (1997) Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet 15(2):146–156PubMedCrossRefGoogle Scholar
  64. Moore GL, Chen H et al (2010) Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions. MAbs 2(2):181–189PubMedCrossRefGoogle Scholar
  65. Muyldermans S, Atarhouch T et al (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7(9):1129–1135PubMedCrossRefGoogle Scholar
  66. O’Connell D, Becerril B et al (2002) Phage versus phagemid libraries for generation of human monoclonal antibodies. J Mol Biol 321(1):49–56PubMedCrossRefGoogle Scholar
  67. Petkova SB, Akilesh S et al (2006) Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease. Int Immunol 18(12):1759–1769PubMedCrossRefGoogle Scholar
  68. Rajpal A, Beyaz N et al (2005) A general method for greatly improving the affinity of antibodies by using combinatorial libraries. Proc Natl Acad Sci U S A 102(24):8466–8471PubMedCrossRefGoogle Scholar
  69. Rathanaswami P, Roalstad S et al (2005) Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8. Biochem Biophys Res Commun 334(4):1004–1013PubMedCrossRefGoogle Scholar
  70. Riechmann L, Muyldermans S (1999) Single domain antibodies: comparison of camel VH and camelised human VH domains. J Immunol Methods 231(1–2):25–38PubMedCrossRefGoogle Scholar
  71. Scott JK, Smith GP (1990) Searching for peptide ligands with an epitope library. Science 249(4967):386–390PubMedCrossRefGoogle Scholar
  72. Shi L, Wheeler JC et al (2010) De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins. J Mol Biol 397(2):385–396PubMedCrossRefGoogle Scholar
  73. Shields RL, Lai J et al (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277(30):26733–26740PubMedCrossRefGoogle Scholar
  74. Shields RL, Namenuk AK et al (2001) High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R. J Biol Chem 276(9):6591–6604PubMedCrossRefGoogle Scholar
  75. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317PubMedCrossRefGoogle Scholar
  76. Soderlind E, Strandberg L et al (2000) Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat Biotechnol 18(8):852–856PubMedCrossRefGoogle Scholar
  77. Stavenhagen JB, Gorlatov S et al (2007) Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcgamma receptors. Cancer Res 67(18):8882–8890PubMedCrossRefGoogle Scholar
  78. Suzuki I, Pfister L et al (1995) Representation of rearranged VH gene segments in the human adult antibody repertoire. J Immunol 154(8):3902–3911PubMedGoogle Scholar
  79. Tomizuka K, Shinohara T et al (2000) Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci U S A 97(2):722–727PubMedCrossRefGoogle Scholar
  80. Tomizuka K, Yoshida H et al (1997) Functional expression and germline transmission of a human chromosome fragment in chimaeric mice. Nat Genet 16(2):133–143PubMedCrossRefGoogle Scholar
  81. Tornetta M, Baker S et al (2010) Antibody Fab display and selection through fusion to the pIX coat protein of filamentous phage. J Immunol Methods 360(1–2):39–46PubMedCrossRefGoogle Scholar
  82. Vettermann C, Herrmann K et al (2006) Powered by pairing: the surrogate light chain amplifies immunoglobulin heavy chain signaling and pre-selects the antibody repertoire. Semin Immunol 18(1):44–55PubMedCrossRefGoogle Scholar
  83. Wang L, Jackson WC et al (2004) Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc Natl Acad Sci U S A 101(48):16745–16749PubMedCrossRefGoogle Scholar
  84. Ward ES, Gussow D et al (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341(6242):544–546PubMedCrossRefGoogle Scholar
  85. Wiberg FC, Rasmussen SK et al (2006) Production of target-specific recombinant human polyclonal antibodies in mammalian cells. Biotechnol Bioeng 94(2):396–405PubMedCrossRefGoogle Scholar
  86. Xu L, Estelles A et al (2010) Surrobodies with functional tails. J Mol Biol 397(1):352–360PubMedCrossRefGoogle Scholar
  87. Xu L, Yee H et al (2008) Combinatorial surrobody libraries. Proc Natl Acad Sci U S A 105(31):10756–10761PubMedCrossRefGoogle Scholar
  88. Yamada M, Wasserman R et al (1991) Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J Exp Med 173(2):395–407PubMedCrossRefGoogle Scholar
  89. Yamagami T, ten Boekel E et al (1999) Four of five RAG-expressing JCkappa-/- small pre-BII cells have no L chain gene rearrangements: detection by high-efficiency single cell PCR. Immunity 11(3):309–316PubMedCrossRefGoogle Scholar
  90. Zahnd C, Spinelli S et al (2004) Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity. J Biol Chem 279(18):18870–18877PubMedCrossRefGoogle Scholar
  91. Zalevsky J, Chamberlain AK et al (2010) Enhanced antibody half-life improves in vivo activity. Nat Biotechnol 28(2):157–159PubMedCrossRefGoogle Scholar
  92. Zebedee SL, Barbas CF 3rd et al (1992) Human combinatorial antibody libraries to hepatitis B surface antigen. Proc Natl Acad Sci U S A 89(8):3175–3179PubMedCrossRefGoogle Scholar
  93. Zhou C, Jacobsen FW et al (2010) Development of a novel mammalian cell surface antibody display platform. MAbs 2(5):508–518PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Ramesh R. Bhatt
    • 1
  • John S. Haurum
    • 2
  • C. Geoffrey Davis
    • 3
  1. 1.Sea Lane BiotechnologiesMountain ViewUSA
  2. 2.ImClone Systems, A wholly-owned subsidiary of Eli Lilly and CompanyNew YorkUSA
  3. 3.Angelica Therapeutics IncAuburnUSA

Personalised recommendations