Research Review | Published:

Engineering antibody Fv fragments for cancer detection and therapy: Bisulfide-stabilized Fv fragments

Nature Biotechnology volume 14, pages 12391245 (1996) | Download Citation

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Abstract

Disulfide-stabilized Fv fragments of antibodies (dsFv) are molecules in which the VH-VL heterodimer is stabilized by an interchain disulfide bond engineered between structurally conserved framework positions distant from complementarity-determining regions (CDRs). This method of stabilization is applicable for the stabilization of many antibody Fvs and has also been applied to a T-cell receptor Fv. A summary of the design strategy, and the construction and production of various dsFvs and dsFv-fusion proteins is presented. Included in the discussion are the biochemical features of dsFvs in comparison with scFvs, the effect of disulfide stabilization on Fv binding and activity, and various applications of dsFvs and dsFv-immunotoxins for tumor imaging and the treatment of solid tumors in animal models.

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References

  1. 1.

    and 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497.

  2. 2.

    , , and 1993. Immunotoxins: magic bullets or misguided missiles? Trends Pharmacol Sci. 14: 148–54.

  3. 3.

    1993. Immunotoxin therapy of cancer. Oncology 7: 69–86.

  4. 4.

    and 1994. Immunotoxins against cancer. Biochem. Biophy. Acta. 1198: 27–45.

  5. 5.

    and 1991. ecombinant toxins for cancer treatment. Science 254: 1173–1137.

  6. 6.

    , , and 1992. Recombinant toxins as novel therapeutic agents. Annu. Rev. Biochem. 61: 331–354.

  7. 7.

    , , , , , , et al. 1988. Single-chain antigen binding proteins. Science 242: 423–426.

  8. 8.

    , , , , , , et al. 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-dogoxigenin single-chain Fv analogue produced in Escherichia coli . Proc. Natl. Acad. Sci. USA 85: 5879–5883.

  9. 9.

    and 1988. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli . Science 240: 1038–1041.

  10. 10.

    , , , and 1994. Making antibodies by phage display technology. Ann. Rev. Immunol. 12: 433–455.

  11. 11.

    1995. Synthetic human antibodies. Nature Medicine 1: 837–839.

  12. 12.

    and 1994. Human antibodies from combinatorial libraries. Adv. Immunol. 57: 191–280.

  13. 13.

    and 1995. Single-chain Fvs. FASEB J. 9: 73–80.

  14. 14.

    , , , , and 1993. A recombinant immunotoxin containing a bisulfide-stabilized Fv fragment. Proc. Natl. Acad. Sci. USA 90: 7538–7542.

  15. 15.

    , , , , , and 1994. Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulflde-stabilized Fv. Protein Eng. 7: 697–704.

  16. 16.

    , , , and 1986. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0 Angstrom. Proc. Natl. Acad. Sci USA 83: 1320–1324.

  17. 17.

    and 1987. Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and show functional homology with glutamic acid 148 of diphtheria toxin. J. Biol. Chem. 262: 8707–8711.

  18. 18.

    , , and 1991. Analysis of Pseudomonas exotoxin activation and conformational changes by using monoclonal antibodies as probes. Infect. Immun. 59: 407–414.

  19. 19.

    and 1989. Pseudomonas exotoxin: chimeric toxins. J. Biol. Chem. 264: 15157–15160.

  20. 20.

    , , , , , , et al. 1991. Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol. 203: 46–88.

  21. 21.

    and 1991. Single-chain Fv proteins and their fusion proteins. Methods: A companion to Methods Enzymol. 2: 97–105.

  22. 22.

    , , , , and 1991. B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Sci. USA 88: 8616–8820.

  23. 23.

    and 1995. Characterization of B1(Fv)-PE38 and B1(dsFv)-PE38: single-chain and disulfide Fv-immunotoxins with increased activity which cause complete remissions of established human carcinoma xenografts in nude mice. Clin. Cancer. Res. 1: 1023–1029.

  24. 24.

    , , , , , , et al. 1993. BR96 sFv-PE30, a potent single-chain immunotoxin that selectively kills carcinoma cells. Cancer Res. 53: 334–339

  25. 25.

    , , , , , and 1991. Characterization of monoclonal antibodies B1 and B3 that react with mucinous adenocarcinomas. Cancer Res. 51: 3781–3787.

  26. 26.

    , , , , , and 1989. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin.Nature 339: 394–397.

  27. 27.

    , , , , and 1992. Recombinant anti-erbB2 immunotoxins containing Pseudomonas exotoxin. Proc. Natl. Acad. Sci. USA 89: 5867–5871.

  28. 28.

    , , , , and 1992. Selective inhibition of tumor cell growth by a recombinant single-chain antibody-toxin specific for the erbB-2 receptor. Cancer Res. 52: 6310–6317.

  29. 29.

    , , and 1994. Design of interchain disulfide bonds in the framework region of the Fv of the monoclonal antibody B3. Proteins: Structure, Function and Genetics 19: 35–47.

  30. 30.

    , , , , and 1991. Sequences of proteins of immunological interest. 5th ed. U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health. NIH publication No. 91-3242.

  31. 31.

    , , , and 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29: 1362–1367.

  32. 32.

    , , , , , , , et al.1992. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. USA 89: 4285–4289.

  33. 33.

    , , , , , , et al. 1995. Development of a humanized disulflde-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug. Cancer Res. 55: 63–70.

  34. 34.

    , , , , and 1995. Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulfide bond. FEBS Lett. 377: 135–139.

  35. 35.

    and 1995. Recombinant immunotoxins: from basic research to cancer therapy. ImmunoMethods. 8: 143–156.

  36. 36.

    , , , , , and 1994. Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions. Biochemistry 33: 5451–5459.

  37. 37.

    1985. Rapid and efficient site-directed mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82: 488–492.

  38. 38.

    and 1986. Use of bacteriophage T7 polymerase to direct selective expression of cloned gene. J. Mol. Biol. 189: 113–130.

  39. 39.

    , , and 1992. A method for increasing the yield of property folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal. Biochem. 205: 263–270.

  40. 40.

    , , , , and 1994. Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulflde-stabilized Fv fragment. Cancer Res. 54: 2714–2718.

  41. 41.

    , , , and 1995. Administration of disulfide-stabilized Fv-immunotoxins B1 (dsFv)-PE38 and B3(dsFv)-PE38 by continuous infusion increases their efficacy in curing large tumor xenografts in nude mice. Int. J. Cancer 62: 351–355.

  42. 42.

    , , , , and 1995. Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog. Mol. Immunol. 32: 249–258.

  43. 43.

    , , , and 1995. Phage display of disulfide-stabilized Fv fragments. J. Immunol. Method 182: 41–50.

  44. 44.

    , , , , , , et al. 1995. Construction of a disulfide stabilized TCR Fv indicates that antibody and TCR Fv frameworks are very similar in structure. Immunity 2: 281–287.

  45. 45.

    , , , and 1995. Crystal structure of the beta chain of a T cell antigen receptor. Science 267: 1984–1987.

  46. 46.

    and 1996. Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer. Clin. Cancer Res. 2: 245–252.

  47. 47.

    , , , , , , and 1994. Improved binding and antjtumor activity of a lecombinant antJ-erbB2 immunotoxin by disulfide stabilization of the Fv foment. J. Biol. Chem. 269: 18327–18331.

  48. 48.

    and 1988. Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: significance of elevated interstitial pressure. Cancer Res. 48: 7022–7033.

  49. 49.

    1992. Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer Res. 50: 814s–819s.

  50. 50.

    , , , and 1992. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res. 52: 3402–3408.

  51. 51.

    , , , and 1996. Recombinant single-chain and disulfide-stabilized Fv immunotoxins that cause complete regression of human colon cancer xenografl in nude mice. Int. J. Cancer. 67: 113–123.

  52. 52.

    and 1995. Cloning expression and characterization of the Fv fragments of the anticarbohydrate monoclonal antibodies B1 and B5 as single-chain immunotoxins. Protein Eng. 7: 1509–1515.

  53. 53.

    and 1996. Recombinant immunotoxin containing a disulfide-stabilized Fv directed at erbB2 that does not require proteolytic activation. Biochemistry 35: 2872–2877.

  54. 54.

    , , , , , and 1993. Recombinant toxins containing the variable domains of the anti-Tac monoclonal antibody to the IL2 receptor kill malignant cells from patients with chronic lymphocytic leukemia. Blood 80: 2344–2352.

  55. 55.

    , , , , , and 1990. The recombinant immunotoxin anti-Tac(Fv)-PE40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia. Proc. Natl. Acad. Sci. USA 87: 8291–8295.

  56. 56.

    , , , , and 1994. Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interieukin-2 receptor-expressing human carcinoma. Blood 83: 426–434.

  57. 57.

    and 1994. Recombinant single-chain immunotoxins against T and B cell leukemias. Leukemia & Lymphoma 13: 1–10.

  58. 58.

    , , , and 1996. Preclinical characterization of e23(dsFv)-PE38, a recombinant disulfide-stabilized Fv immunotoxin directed at erbB2. Clin. Cancer Res. Submitted.

  59. 59.

    and 1996. Improved antitumor activity of a recombinant anti-LewisY immunotoxin not requiring proteolytic activation. Proc. Natl. Acad. Sci. USA 93: 974–978.

  60. 60.

    , , , and 1992. The multichain inter-leukin-2 receptor a target for immunotherapy. Ann. Intern. Med. 116: 148–160.

  61. 61.

    , , and 1995 Rapid and specific uptake of anti-Tac disulfide-stabilized Fv by interleukin-2 receptor bearing tumors. Cancer Res. 55: 318–323.

  62. 62.

    , , , , , , et al. 1995. Biodistribution of 18F- and 125I-labeled anti-Tac disulfide-stabilized Fv fragments in nude mice with interieukin 22 receptor-positive tumor xenografts. Cancer Res. 55: 5323–5329.

  63. 63.

    , , , , and 1993. A bacterially expressed single-chain Fv construct from the 2B4 T-cell receptor. Proc. Natl. Acad. Sci. USA 90: 3830–3834.

  64. 64.

    , , , , , and 1992. Characterization of a single-chain T-cell receptor expressed in E. coli . Proc. Natl. Acad. Sci. USA 89: 4759–4763.

  65. 65.

    , , , , , , et al. 1991. A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties. Proc. Natl. Acad. Sci. USA 88: 8646–8650.

  66. 66.

    1992. Secretion of T-cell receptor fragments from recombinant E. colicells. J. Mol. Biol. 224: 885–890.

  67. 67.

    and 1994. Correctly folded T-cell receptor fragments in the periplasm of E. coli . J. Mol. Biol.242: 655–669.

  68. 68.

    , , , , , , et al. 1992. Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor. J. Immunol. 149: 2810–2815.

  69. 69.

    , , , and 1990. A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin. Proc. Natl. Acad. Sci. USA 87: 9491–9494.

  70. 70.

    , , , and 1991. Single chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv). Mol. Cell Biol. 11: 2200–2205.

  71. 71.

    , , , , , , et al. 1989. Antitumor activity in mice of an immunotoxin made with anti-transferrin receptor and a recombinant form of Pseudomonasexotoxin. Proc. Natl. Acad. Sci. USA 86: 8545–8549.

  72. 72.

    , , , , and 1993. A recombinant immunotoxin active on prostate cancer cells composed of the Fv-region of MAb PR1 and a truncated form of Pseudomonasexotoxin. Proc. Natl. Acad. Sci USA 90: 547–551.

  73. 73.

    , , , and 1990. Pseudomonasexotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc. Natl. Acad. Sci. USA 87: 308–312.

  74. 74.

    , , , and 1991. Increased cytotoxic activity of Pseudomonasexotoxin and two chimeric toxins ending in KDEL. J. Biol. Chem. 266: 17376–17381.

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Author notes

    • Ira Pastan

    Corresponding author.

Affiliations

  1. Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 4E16, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255.

    • Yoram Reiter
    • , Ulrich Brinkmann
    • , Byungkook Lee
    •  & Ira Pastan

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https://doi.org/10.1038/nbt1096-1239