Biological activity and in vivo clearance of antitumor antibody/cytokine fusion proteins.
Several human cytokines including IL-2, GM-CSF, and tumor necrosis factors alpha and beta were engineered as fusion proteins to the carboxyl terminus of a chimeric anti-ganglioside antibody, ch14.18, and expressed in transfected hybridoma cells. All of the fusion proteins were expressed at high levels and were easily purified by affinity or ion-exchange chromatography from culture supernatants. The effect of fusion on antigen binding activity was tested and found to vary with the particular cytokine. No significant decreases in antigen binding were observed, and fusion of IL-2 had the greatest positive effect in a direct antigen binding assay. All fusion proteins maintained normal levels of biological activity except for GM-CSF, which was approximately 20% active, compared to recombinant GM-CSF produced in bacteria. The clearance of the fusion proteins was examined in normal Balb/c mice after intraperitoneal injection or in athymic (nu/nu) mice after intravenous injection and was generally quite rapid, relative to ch14.18. This was mainly due to a very rapid initial clearance rate (alpha phase) since the half-lives of the beta phase of the fusion proteins (about 30 h) were comparable to that of the free antibody (about 58 h). These results demonstrate that biologically active antibody/cytokine fusion proteins can be constructed by genetic engineering. Their relatively rapid clearance may require constant infusion rather than bolus injection in order to achieve clinical efficacy.
Gillies
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SD
Abbott Biotech, Inc, Needham Heights, Massachusetts 02194.
Young
D
D
Lo
K M
KM
Roberts
S
S
D000818:Animals; D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology; D016207:Cytokines / Q000235:genetics / Q000493:pharmacokinetics* / Q000494:pharmacology*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000378:metabolism* / Q000494:pharmacology*; D008545:Melanoma / Q000188:drug therapy / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology
D000906:Antibodies; D016207:Cytokines; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D012967:Sodium Dodecyl Sulfate; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D010450:Endopeptidases