Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Apr 15;89(8):3576–3580. doi: 10.1073/pnas.89.8.3576

In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.

H Gram 1, L A Marconi 1, C F Barbas 3rd 1, T A Collet 1, R A Lerner 1, A S Kang 1
PMCID: PMC48911  PMID: 1565653

Abstract

We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin mu and kappa light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 10(4)-10(5) M-1) binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the mono-valent phage display vector pComb3 was performed by error-prone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.

Full text

PDF
3576

Images in this article

3578Image
on p.3578
3579Image
on p.3579

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barbas C. F., 3rd, Kang A. S., Lerner R. A., Benkovic S. J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7978–7982. doi: 10.1073/pnas.88.18.7978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berek C., Milstein C. Mutation drift and repertoire shift in the maturation of the immune response. Immunol Rev. 1987 Apr;96:23–41. doi: 10.1111/j.1600-065x.1987.tb00507.x. [DOI] [PubMed] [Google Scholar]
  3. Better M., Chang C. P., Robinson R. R., Horwitz A. H. Escherichia coli secretion of an active chimeric antibody fragment. Science. 1988 May 20;240(4855):1041–1043. doi: 10.1126/science.3285471. [DOI] [PubMed] [Google Scholar]
  4. 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., Whitlow M. Single-chain antigen-binding proteins. Science. 1988 Oct 21;242(4877):423–426. doi: 10.1126/science.3140379. [DOI] [PubMed] [Google Scholar]
  5. Burton D. R., Barbas C. F., 3rd, Persson M. A., Koenig S., Chanock R. M., Lerner R. A. 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. 1991 Nov 15;88(22):10134–10137. doi: 10.1073/pnas.88.22.10134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caton A. J., Koprowski H. Influenza virus hemagglutinin-specific antibodies isolated from a combinatorial expression library are closely related to the immune response of the donor. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6450–6454. doi: 10.1073/pnas.87.16.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chiang Y. L., Sheng-Dong R., Brow M. A., Larrick J. W. Direct cDNA cloning of the rearranged immunoglobulin variable region. Biotechniques. 1989 Apr;7(4):360–366. [PubMed] [Google Scholar]
  8. Clackson T., Hoogenboom H. R., Griffiths A. D., Winter G. Making antibody fragments using phage display libraries. Nature. 1991 Aug 15;352(6336):624–628. doi: 10.1038/352624a0. [DOI] [PubMed] [Google Scholar]
  9. Decker D. J., Boyle N. E., Koziol J. A., Klinman N. R. The expression of the Ig H chain repertoire in developing bone marrow B lineage cells. J Immunol. 1991 Jan 1;146(1):350–361. [PubMed] [Google Scholar]
  10. Deverson E., Berek C., Taussig M., Feinstein A. Monoclonal BALB/c anti-progesterone antibodies use family IX variable region heavy chain genes. Eur J Immunol. 1987 Jan;17(1):9–13. doi: 10.1002/eji.1830170103. [DOI] [PubMed] [Google Scholar]
  11. Diamond B., Scharff M. D. Somatic mutation of the T15 heavy chain gives rise to an antibody with autoantibody specificity. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5841–5844. doi: 10.1073/pnas.81.18.5841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ewulonu U. K., Nell L. J., Thomas J. W. VH and VL gene usage by murine IgG antibodies that bind autologous insulin. J Immunol. 1990 Apr 15;144(8):3091–3098. [PubMed] [Google Scholar]
  13. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fowler R. G., Schaaper R. M., Glickman B. W. Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3'----5' exonuclease (proofreading) activity. J Bacteriol. 1986 Jul;167(1):130–137. doi: 10.1128/jb.167.1.130-137.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Griffiths G. M., Berek C., Kaartinen M., Milstein C. Somatic mutation and the maturation of immune response to 2-phenyl oxazolone. Nature. 1984 Nov 15;312(5991):271–275. doi: 10.1038/312271a0. [DOI] [PubMed] [Google Scholar]
  16. Hartman A. B., Rudikoff S. VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules. EMBO J. 1984 Dec 1;3(12):3023–3030. doi: 10.1002/j.1460-2075.1984.tb02249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huse W. D., Sastry L., Iverson S. A., Kang A. S., Alting-Mees M., Burton D. R., Benkovic S. J., Lerner R. A. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science. 1989 Dec 8;246(4935):1275–1281. doi: 10.1126/science.2531466. [DOI] [PubMed] [Google Scholar]
  18. Kang A. S., Barbas C. F., Janda K. D., Benkovic S. J., Lerner R. A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4363–4366. doi: 10.1073/pnas.88.10.4363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kang A. S., Jones T. M., Burton D. R. Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11120–11123. doi: 10.1073/pnas.88.24.11120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klein D., Nietupski J., Sirlin S., Stavnezer J. I.29 lymphoma cells express a nonmutated VH gene before and after H chain switch. J Immunol. 1988 Mar 1;140(5):1676–1684. [PubMed] [Google Scholar]
  21. Kocks C., Rajewsky K. Stable expression and somatic hypermutation of antibody V regions in B-cell developmental pathways. Annu Rev Immunol. 1989;7:537–559. doi: 10.1146/annurev.iy.07.040189.002541. [DOI] [PubMed] [Google Scholar]
  22. Larimer F. W., Mural R. J., Soper T. S. Versatile protein engineering vectors for mutagenesis, expression and hybrid enzyme formation. Protein Eng. 1990 Jan;3(3):227–231. doi: 10.1093/protein/3.3.227. [DOI] [PubMed] [Google Scholar]
  23. Malipiero U. V., Levy N. S., Gearhart P. J. Somatic mutation in anti-phosphorylcholine antibodies. Immunol Rev. 1987 Apr;96:59–74. doi: 10.1111/j.1600-065x.1987.tb00509.x. [DOI] [PubMed] [Google Scholar]
  24. Marks J. D., Hoogenboom H. R., Bonnert T. P., McCafferty J., Griffiths A. D., Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991 Dec 5;222(3):581–597. doi: 10.1016/0022-2836(91)90498-u. [DOI] [PubMed] [Google Scholar]
  25. McCafferty J., Griffiths A. D., Winter G., Chiswell D. J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990 Dec 6;348(6301):552–554. doi: 10.1038/348552a0. [DOI] [PubMed] [Google Scholar]
  26. Nakane P. K. Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J Histochem Cytochem. 1968 Sep;16(9):557–560. doi: 10.1177/16.9.557. [DOI] [PubMed] [Google Scholar]
  27. Ollo R., Auffray C., Sikorav J. L., Rougeon F. Mouse heavy chain variable regions: nucleotide sequence of a germ-line VH gene segment. Nucleic Acids Res. 1981 Aug 25;9(16):4099–4109. doi: 10.1093/nar/9.16.4099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Orlandi R., Güssow D. H., Jones P. T., Winter G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 May;86(10):3833–3837. doi: 10.1073/pnas.86.10.3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Parmley S. F., Smith G. P. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene. 1988 Dec 20;73(2):305–318. doi: 10.1016/0378-1119(88)90495-7. [DOI] [PubMed] [Google Scholar]
  30. Persson M. A., Caothien R. H., Burton D. R. Generation of diverse high-affinity human monoclonal antibodies by repertoire cloning. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2432–2436. doi: 10.1073/pnas.88.6.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rath S., Stanley C. M., Steward M. W. An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity. J Immunol Methods. 1988 Feb 10;106(2):245–249. doi: 10.1016/0022-1759(88)90204-9. [DOI] [PubMed] [Google Scholar]
  32. Sastry L., Alting-Mees M., Huse W. D., Short J. M., Sorge J. A., Hay B. N., Janda K. D., Benkovic S. J., Lerner R. A. Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5728–5732. doi: 10.1073/pnas.86.15.5728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Seidman J. G., Max E. E., Leder P. A kappa-immunoglobulin gene is formed by site-specific recombination without further somatic mutation. Nature. 1979 Aug 2;280(5721):370–375. doi: 10.1038/280370a0. [DOI] [PubMed] [Google Scholar]
  34. Skerra A., Plückthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988 May 20;240(4855):1038–1041. doi: 10.1126/science.3285470. [DOI] [PubMed] [Google Scholar]
  35. Stura E. A., Arevalo J. H., Feinstein A., Heap R. B., Taussig M. J., Wilson I. A. Analysis of an anti-progesterone antibody: variable crystal morphology of the Fab' and steroid-Fab' complexes. Immunology. 1987 Dec;62(4):511–521. [PMC free article] [PubMed] [Google Scholar]
  36. Ward E. S., Güssow D., Griffiths A. D., Jones P. T., Winter G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature. 1989 Oct 12;341(6242):544–546. doi: 10.1038/341544a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES