The potential immunogenicity of human insulin and insulin analogues evaluated in a transgenic mouse model.
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.
Ottesen
J L
JL
Biopharmaceuticals Division, Novo Nordisk, Gentofte, Denmark.
Nilsson
P
P
Jami
J
J
Weilguny
D
D
Dührkop
M
M
Bucchini
D
D
Havelund
S
S
Fogh
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001483:Base Sequence; D002417:Cattle; D003433:Crosses, Genetic; D017931:DNA Primers; D005260:Female; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D007330:Insulin Antibodies / Q000096:biosynthesis*; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013552:Swine
D017931:DNA Primers; D007328:Insulin; D007330:Insulin Antibodies; D046911:Macromolecular Substances; D011384:Proinsulin