Summary
Transgenic mice with tissue-specific expression of the human insulin gene in the beta cells of the pancreas do not produce insulin-specific antibodies when injected with human insulin. Tolerant transgenic mice injected with human or porcine insulin reflect the clinical situation. When injected with bovine insulin the transgenic mice produce antibodies. The potential immunogenicity of 12 recombinant human insulin analogues has been tested in this transgenic model. The analogues were designed either to prevent hexamer formation or to improve chemical stability or both. The analogues have amino acid substitutions or deletions at residue 8, 10 and 21 in the A-chain and residue 3, 9, 27 and 28 in the B-chain. The results show that substitution of single amino acids in the A-chain loop of human insulin for the corresponding amino acids in bovine insulin at residues A8 or A10 is sufficient to elicit an antibody response in responder mice. Only human insulin analogues with substitutions at residues 8 or 10 in the A-chain elicit antibody formation in the transgenic mice, whereas non-transgenic control groups respond to insulin and all analogues. Antibodies developed against the human insulin analogues are cross reactive with recombinant human insulin. Antibodies developed against an immunogenic analogue could therefore neutralize both the analogue and the native insulin and thereby aggravate the patient's condition. This transgenic mouse immunogenicity model should be useful as an in vivo model to map immunogenic areas of recombinant proteins.
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Abbreviations
- MHC:
-
Major histocompatibility complex
- PCR:
-
polymerase chain reaction
- OD:
-
optical density
- htPA:
-
human tissue plasminogen activator
- RIA:
-
radioimmunoassay
References
Reeves WG (1983) Insulin antibody determination: theoretical and practical considerations. Diabetologia 24: 399–403
Stewart TA, Hollingshead PG, Pitts SL, Chang R, Martin LE, Oakley H (1989) Transgenic mice as a model to test the immunogenicity of proteins altered by site-specific mutagenesis. Mol Biol Med 6: 275–281
Keck K, Momayezi M (1980) Genetic regulation of the immune response of mice to insulins of different species. In: Brandenburg D, Wallmer A (eds) Insulin, chemistry, structure and function of insulin and related hormones. Walther de Gruyter, Berlin, pp 551–558
Keck K (1977) Ir gene control of carrier recognition. III. Cooperative recognititon of two or more carrier determinants of different species. Eur J Immunol 7: 811–816
Keck K (1975) Ir-gene control of immunogenicity of insulin and A-chain loop as a carrier determinant. Nature (Lond) 254: 78–79
Kapp JA, Strayer DS (1978) H-2 linked Ir gene control of antibody responses to porcine insulin. I. Development of insulin-specific antibodies in some but not all nonresponder strains injected with proinsulin. J Immunol 121: 978–982
Hanahan D (1989) Transgenic mice as probes into complex systems. Science 246: 1265–1275
Adams TE (1990) Tolerance to self-antigens in transgenic mice. Mol Biol Med 7: 341–357
Miller JFAP, Morahan G, Allison J, Hoffmann M (1991) A transgenic approach to the study of peripheral T-cell tolerance. Immunol Rev 122: 103–116
Lo D, Burkley LC, Flavell RA, Palmiter RD, Brinster RL (1990) Antigen presentation in MHC class II transgenic mice: stimulation versus tolerization. Immunol Rev 117: 121–134
Basten A, Brink R, Peake P et al. (1991) Self tolerance in the B-cell repertoire. Immunol Rev 122: 5–19
Lo D, Freedman J, Hesse S, Brinster RL, Sherman L (1991) Peripheral tolerance in transgenic mice: tolerance to class II MHC and non-MHC transgene antigens. Immunol Rev 122: 87–102
Lipes MA, Eisenbarth GS (1990) Transgenic mouse models of type 1 diabetes. Diabetes 39: 879–884
Bucchini D, Ripoche MA, Stinnakre MG et al. (1986) Pancreatic expression of human insulin gene in transgenic mice. Proc Natl Acad Sci USA 83: 2511–2515
Michalova K, Bucchini D, Ripoche MA, Pictet R, Jami J (1988) Chromosome localization of the human insulin gene in transgenic mouse lines. Hum Genet 80: 247–252
Bucchini D, Madsen O, Desbois P, Jami J, Pictet R (1989) B islet cells of pancreas are the site of expression of the human insulin gene in transgenic mice. Exp Cell Res 180: 467–474
Fromont-Racine M, Bucchini D, Madsen O et al. (1990) Effect of 5′-flanking sequence deletions on expression of the human insulin gene in transgenic mice. Mol Endo 4: 669–677
Laird PW, Zijderveld A, Linders K, Rudnicki MA, Janisch R, Berns A (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19: 4293
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd. edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum Anal Biochem 137: 266–267
Bell GL, Pictet RL, Rutter WJ, Cordell B, Tischer E, Goodman HM (1989) Sequence of the human insulin gene. Nature 284: 26–32
Saiki RK (1990) Amplification of genomic DNA. In: Innis MA, Gelfand DH, Sniskky JJ, White TJ (eds) PCR protocols. A guide to methods and applications. Academic Press, San Diego, pp 13–20
Markussen J, Diers I, Hougaard P et al. (1988) Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30. Protein Eng 2: 157–166
Brange J (1992) Chemical stability of insulin. 4. Mechanisms and kinetics of chemical transformations in pharmaceutical formulation. Acta Pharm Nord 4: 209–222
Brange J, Ribel U, Hansen JF et al. (1988) Monomeric insulins obtained by protein engineering and their medical implications. Nature 333: 679–682
Balschmidt P, Brange J (1992) Fast acting human insulin analogues with a single amino acid deletion in the B-chain (Abstract). Diabetologia 35 [Suppl 1]: A4
Whiteley PJ, Lake JP, Selden RF, Kapp JA (1989) Tolerance induced by physiological levels of secreted proteins in transgenic mice expressing human insulin. J Clin Invest 84: 1550–1554
Dayhoff MO (1972) Atlas of protein sequence and structure, vol. 5. The National Medical Research Foundation, Washington
Hansen B (1981) Immunogenicity of insulin as a function of species and other factors. In: Gueriguian JL (ed) Insulins, growth hormone, and recombinant DNA technology. Raven Press, New York, pp 55–70
Kasama T, Iwata Y, Oshiro K et al. (1981) Antigenicity of desamido-insulin and monocomponent insulin. Diabetologia 21: 65–69
Schlichtkrull J, Brange J, Christiansen AH, Hallund O, Heding LG, JØrgensen KH (1972) Clinical aspects of insulin-antigenicity. Diabetes 21 [Suppl 2]: 649–656
Root MA, Chance RE, Galloway JA (1972) Immunogenicity of insulin. Diabetes 21 [Suppl 2]: 657–660
Brange J, Owens DR, Kang S, VØlund A (1990) Monomeric insulins and their experimental and clinical implications. Diabetes Care 13: 923–954
Parkar BA, Reeves WG (1989) In vitro priming of human lymphocytes to heterologous insulins. J Immunol Meth 120: 159–165
Schroer JA, Inman JK, Thomas JW, Rosenthal AS (1979) H-2-linked Ir gene control of antibody responses to insulin. I. Anti-insulin plaque-forming cell primary responses. J Immunol 123: 670–675
Jensen PE, Kapp JA (1984) Regulatory mechanisms of the immune response to heterologous insulins. I. Development and regulation of PFC responses in vitro. Cell Immunol 87: 73–84
Marshall MO, Heding LG, Villumsen J et al. (1988) Development of insulin antibodies, metabolic control and B-cell function in newly diagnosed insulin dependent diabetic children treated with monocomponent human insulin or monocomponent porcine insulin. Diabetes Res 9: 169–175
Zuppinger K, Aebi C, Fankhauser S et al. (1987) Comparison of human and porcine insulin therapies in children with newly diagnosed diabetes mellitus. Diabetologia 30: 912–915
Heding LG, Marshall MO, Persson B et al. (1984) Immunogenicity of monocomponent human and porcine insulin in newly diagnosed type 1 (insulin-dependent) diabetic children. Diabetologia 27: 96–98
Reeves WG (1986) The immune response to insulin: characterisation and clinical consequences. In: Alberti KGMM, Krall LP (eds) The diabetes annual Vol 2. Elsevier Science Publishers, Amsterdam, pp 81–93
Schernthaner G, Borkenstein M, Fink M, Mayr WR, Menzel J, Schober E (1983) Immunogenicity of human insulin (Novo) or pork monocomponent insulin in HLA-DR-typed insulin-dependent diabetic individuals. Diabetes Care 6: 43–48
Reeves WG (1985) Immunogenicity of insulin of various origins. Neth J Med 28: 43–46
VØlund A, Brange J, Drejer K et al. (1991) In vitro and in vivo potency of insulin analogues designed for clinical use. Diabetic Med 8: 839–847
Whiteley PJ, Kapp JA (1989) Tolerance in transgenic mice to a non-MHC self-protein is not a result of clonal deletion. Progress in Immunol 7: 826–832
Roberts HR, Cromatie R (1984) Overview of inhibitors to factor VIII and IX. In: Hoyer LW (ed) Factor VIII inhibitors. Alan R. Liss, New York, pp 1–18
Capecchi MR (1989) Altering the genome by homologous recombination. Science 244: 1288–1292
Sedivy JM, Joyner AL (1992) Gene targeting. WH Freeman and Company, New York
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Ottesen, J.L., Nilsson, P., Jami, J. et al. The potential immunogenicity of human insulin and insulin analogues evaluated in a transgenic mouse model. Diabetologia 37, 1178–1185 (1994). https://doi.org/10.1007/BF00399790
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DOI: https://doi.org/10.1007/BF00399790