A method for preparing chelate-cytokine conjugates with retention of protein structure, biological activity, and pharmacokinetic properties
| Autor: | Melissa A Woodward, David C. Litzinger, Ralph W. Niven, Wendy Laderach, K Lane Whitcomb, Lloyd D. Ralph, Michael J. Treuheit, Charles M. Ransone |
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| Rok vydání: | 1995 |
| Předmět: |
Molecular Sequence Data
Immunology Protein structure Humans Immunology and Allergy Chelation Amino Acid Sequence Protein secondary structure Chromatography High Pressure Liquid Chelating Agents Chromatography Chemistry Indium Radioisotopes Receptors Interleukin-2 Fast protein liquid chromatography Biological activity Pentetic Acid respiratory system In vitro Interleukin-2 Electrophoresis Polyacrylamide Gel Cytokine receptor Chromatography Liquid Protein Binding circulatory and respiratory physiology Conjugate |
| Zdroj: | Journal of Immunological Methods. 187:151-161 |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/0022-1759(95)00179-e |
| Popis: | 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 (α 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. |
| Databáze: | OpenAIRE |
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