title: A method for preparing chelate-cytokine conjugates with retention of protein structure, biological activity, and pharmacokinetic properties.
abstract: The chelating agent diethylenetriaminepentaacetic acid (DTPA) has been conjugated site-specifically to the N-terminus of recombinant human interleukin-2 (rhIL-2) by reaction with DTPA dianhydride at an initial pH of 6.0, thus demonstrating broader application of the conjugation method previously described for the structurally related cytokine rhG-CSF (Ralph et al., 1995). Purity of the DTPA-rhIL-2 conjugate, isolated by cation-exchange FPLC, and chelation of 111In were revealed by cation-exchange HPLC. Purity of the conjugate as well as chelation of radiometal were also demonstrated by SDS-PAGE and TLC, respectively. The stoichiometric molar ratio of DTPA to protein for the conjugate was approximately 1:1 as determined by TLC and mass spectrometry. Localization of the DTPA moiety was resolved by a peptide mapping procedure. The protein retained > 95% secondary structure (alpha helicity) following the conjugation. Addition of metal induced an approximate 22% loss of secondary structure for the conjugate. The in vitro biological activity of the protein was unaffected by the conjugated DTPA, even with chelated metal. Pharmacokinetic analysis of DTPA-conjugated cytokines, following chelated 111In, showed clearance and pharmacokinetic parameter values comparable to those of the corresponding unmodified cytokine. DTPA-conjugated cytokines may prove useful in cytokine research, and furthermore may represent a novel class of molecules for imaging, diagnosing, and/or treatment of malignancies where the cytokine receptor is overexpressed.
mesh_terms: D000595:Amino Acid Sequence; D002614:Chelating Agents / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007205:Indium Radioisotopes; D007376:Interleukin-2 / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics*; D008969:Molecular Sequence Data; D004369:Pentetic Acid / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015375:Receptors, Interleukin-2 / Q000032:analysis
chemical_list: D002614:Chelating Agents; D007205:Indium Radioisotopes; D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D004369:Pentetic Acid