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By–Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling

Abstract

Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response1. Here we have improved the affinity of one such “primary” antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V–genes (chain shuffling)2 obtained from unimmunized donors. For a human phage antibody for the hapten 2–phenyloxazol–5–one (phOx) (Kd=3.2×10−7 M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15–fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd=1.1×10−9 M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.

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References

  1. 1

    Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferly, J., Griffiths, A.D. and Winter, G. 1991. By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222: 581–597.

    CAS  Article  Google Scholar 

  2. 2

    Clackson, T., Hoogenboom, H.R., Griffiths, A.D. and Winter, G. 1991. Making antibody fragments using phage display libraries. Nature 352: 624–628.

    CAS  Article  Google Scholar 

  3. 3

    Winter, G. and Milstein, C. 1991. Man-made antibodies. Nature 349: 293–299.

    CAS  Article  Google Scholar 

  4. 4

    Jones, P., Dear, P., Foote, J., Neuberger, M. and Winter, G. 1986. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321: 522–525.

    CAS  Article  Google Scholar 

  5. 5

    Riechmann, L., Clark, M., Waldmann, H. and Winter, G. 1988. Reshaping human antibodies for therapy. Nature 332: 323–327.

    CAS  Article  Google Scholar 

  6. 6

    Queen, C., Schneider, W.P., Selick, H.E., Payne, P.W., Landolfi, N.F., Duncan, J.F., Avdalovic, N.M., Levitt, M., Junghans, R.P. and Waldmann, T.A. 1989. A humanized antibody that binds to the interleukin 2 receptor. Proc. Natl. Acad. Sci. USA 86: 10029–10033.

    CAS  Article  Google Scholar 

  7. 7

    Tempest, P.R., Bremner, P., Lambert, M., Taylor, G., Furze, J.M., Carr, F.J., Harris, W.J. 1991. Reshaping a monoclonal antibody to inhibit human respiratory syncytial virus infection in vivo . Bio/Technology 9: 266–272.

    CAS  Google Scholar 

  8. 8

    Gorman, S.D., Clark, M.R., Routledge, F.G., Cobbold, S.P. and Waldmann, H. 1991. Reshaping a therapeutic CD4 antibody. Proc. Natl. Acad. Sci. USA 88: 4181–4185.

    CAS  Article  Google Scholar 

  9. 9

    Hale, G., Dyer, M.J., Clark, M.R., Phillips, J.M., Marcus, R., Riechmann, L., Winter, G. and Waldmann, H. 1988. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAM PATH-1H. Lancet 2: 1394–1399.

    CAS  Article  Google Scholar 

  10. 10

    Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. 1985. Enzymatic amplification of μ -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354.

    CAS  Article  Google Scholar 

  11. 11

    Orlandi, R., Gussow, D.H., Jones, P.T. and Winter, G. 1989. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86: 3833–3837.

    CAS  Article  Google Scholar 

  12. 12

    Huse, W.D., Sastry, L., Iverson, S.A., Kang, A.S., Alting, M.M., Burton, D.R., Benkovic, S.J. and Lerner, R.A. 1989. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246: 1275–1281.

    CAS  Article  Google Scholar 

  13. 13

    Smith, G.P. 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317.

    CAS  Article  Google Scholar 

  14. 14

    Parmley, S.F. and Smith, G.P. 1988. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73: 305–318.

    CAS  Article  Google Scholar 

  15. 15

    McCafferty, J., Griffiths, A.D., Winter, G. and Chiswell, D.J. 1990. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348: 552–554.

    CAS  Article  Google Scholar 

  16. 16

    Burton, D.R., Barbas, C.F., Persson, M.A.A., Chanock, R.M. and Lerner, R.A. 1991. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic individuals. Proc. Natl. Acad. Sci. USA 88: 10134–10137.

    CAS  Article  Google Scholar 

  17. 17

    Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. 1988. Single-chain antigen-binding proteins. Science 242: 423–426.

    CAS  Article  Google Scholar 

  18. 18

    Huston, J.S., Levinson, D., Mudgett, H.M., Tai, M.S., NovotnyJ Margo-lies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. and Oppermann, H. 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli . Proc. Natl. Acad. Sci. USA 85: 5879–5883.

    CAS  Article  Google Scholar 

  19. 19

    Hawkins, R.E., Russell, S.J. and Winter, G. 1992. Selection of phage antibodies by binding affinity: mimicking affinity maturation. J. Mol. Biol. In Press.

  20. 20

    Kang, A.S., Jones, T.M. and Burton, D.R. 1991. Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries. Proc. Natl. Acad. Sci. USA 88: 11120–11123.

    CAS  Article  Google Scholar 

  21. 21

    Hoogenboom, H.R., Griffiths, A.D., Johnson, K.S., Chiswell, D.J., Hudson, P. and Winter, G. 1991. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucl. Acids Res. 19: 4133–4137.

    CAS  Article  Google Scholar 

  22. 22

    Rath, S., Stanley, C.M. and Steward, M.W. 1988. An inhibition enzyme immunosassay for estimating relative antibody affinity and affinity heterogeneity. J. Immunol. Methods 106: 245–249.

    CAS  Article  Google Scholar 

  23. 23

    Jönsson, U., Fägerstam, L., Ivarsson, B., Lundh, K., Löfås, S., Persson, B., Roos, H., Rönnberg, I., Sjölander, S., Stenberg, E., Ståhlberg, R., Urbaniczky, C., Östlin, H. and Malmqvist, M. 1991. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. BioTechniques 11: 620–627.

    PubMed  Google Scholar 

  24. 24

    Jönsson, U. and Malmqvist, M. 1992. Real time biospecific interaction, p. 291–336. In: Advances in Biosensors. Turner (Ed.) JAI Press Ltd. San Diego, CA.

    Google Scholar 

  25. 25

    Chothia, C. and Lesk, A.M. 1987. Canonical structures for the hyperva riable regions of immunoglobulins. J. Mol. Biol. 196: 901–917.

    CAS  Article  Google Scholar 

  26. 26

    Foote, J. and Milstein, C. 1991. Kinetic maturation of an immune response. Nature 352: 530–532.

    CAS  Article  Google Scholar 

  27. 27

    Berek, C., Griffiths, G.M. and Milstein, C. 1985. Molecular events during maturation of the immune response to oxazolone. Nature 316: 412–418.

    CAS  Article  Google Scholar 

  28. 28

    Berek, C. and Milstein, C. 1987. Mutation drift and repertoire shift in the maturation of the immune response. Immunol. Rev. 96 23–41.

    CAS  Article  Google Scholar 

  29. 29

    Garrard, L.J., Yang, M., O'Connell, M.P., Kelley, R.F. and Henner, D.J. 1991. Fab assembly and enrichment in a monovalent phage display system. Bio/Technology 9: 1373–1377.

    CAS  Article  Google Scholar 

  30. 30

    Perelson, A.S. 1989. Immune network theory. Immunol. Rev. 110 5–36.

    CAS  Article  Google Scholar 

  31. 31

    Sharon, J. 1990. Structural correlates of high antibody affinity. Three engineered amino acids substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold. Proc. Natl. Acad. Sci. USA 87: 4814–4817.

    CAS  Article  Google Scholar 

  32. 32

    Sambrook, J., Fritsch, E.F. and Maniatis, T. 1990. Molecular Cloning—A Laboratory Manual. Cold Spring Harbor Laboratory, New York.

    Google Scholar 

  33. 33

    Dower, W.J., Miller, J.F. and Ragsdale, C.W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res. 16: 6127–6145.

    CAS  Article  Google Scholar 

  34. 34

    Gibson, T.J. 1984. Studies on the Epstein-Barr virus genome. Ph.D. Thesis, University of Cambride, UK.

    Google Scholar 

  35. 35

    Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York.

    Google Scholar 

  36. 36

    Tomlinson, I.M., Walter, G., Marks, J.D., Llewelyn, M.B. and Winter, G. 1992. The repertoire of human germline VH sequences reveals fifty groups of VH segments with different hypervariable loops. J. Mol. Biol. In Press.

  37. 37

    Carter, P., Bedouelle, H. and Winter, G. 1985. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucl. Acids Res. 13: 4431–4443.

    CAS  Article  Google Scholar 

  38. 38

    De Bellis, D. and Schwartz, I. 1990. Regulated expression of foreign genes fused to lac: control by glucose levels in growth medium. Nucl. Acids Res. 18 1311.

    CAS  Article  Google Scholar 

  39. 39

    Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.

    CAS  Article  Google Scholar 

  40. 40

    Munro, S. and Pelham, H.R.B. 1986. An Hsp like protein in the ER: Identity with the 78kd glucose regulated protein and immunoglobulin heavy chain binding protein. Cell 46: 291–300.

    CAS  Article  Google Scholar 

  41. 41

    Eisen, H.N. 1964. Determination of antibody affinity for haptens and antigens by means of fluorescence quenching. Meth. Med. Research 10: 115–121.

    CAS  Google Scholar 

  42. 42

    Johnsson, B., Löfås, S. and Lindqvist, G. 1991. Immobilization of proteins to a carboxymethyldextran modified gold surface for BIAcore in surface plasmon resonance. Anal. Biochem. 198: 268–277.

    CAS  Article  Google Scholar 

  43. 43

    Karlsson, R., Michaelsson, A. and Mattsson, L. 1991. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J. Immunol. Methods 145: 229–240.

    CAS  Article  Google Scholar 

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Marks, J., Griffiths, A., Malmqvist, M. et al. By–Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling. Nat Biotechnol 10, 779–783 (1992). https://doi.org/10.1038/nbt0792-779

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